Process for detecting the presence or quantity of enzymatic activity in a sample

ABSTRACT

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to concurrently filed U.S. patentapplication Ser. No. ______, filed Mar. 12, 2002, Rabbani et al., thatapplication being titled “Real-Time Nucleic Acid Detection Processes andCompositions.” The contents of the aforementioned Ser. No. ______ arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the field of labeling chemistryincluding labeling reagents, processes for target labeling, labeledtargets, processes for preparing labeling reagents, and the like. Thisinvention also relates to the use of such compositions and processes inother processes for nucleic acid and enzymatic activity determinationsand analyses.

[0003] All patents, patent applications, patent publications, scientificarticles and the like, cited or identified in this application arehereby incorporated by reference in their entirety in order to describemore fully the state of the art to which the present invention pertains.

BACKGROUND OF THE INVENTION

[0004] For purposes of organization, this background has been dividedinto seven parts as follows:

[0005] (1) Reactive Groups of Labeling Reagents

[0006] (2) Linker Arms for Connecting Labels to Targets

[0007] (3) Porphyrin Fluorescent Dyes as Labels

[0008] (4) Alterations in Fluorescent Properties

[0009] (5) Fluorescent Intercalators

[0010] (6) Chemiluminescence

[0011] (6) Real Time Detection through Fluorescence

[0012] (7) Primer Binding Sequences in Analytes

[0013] (1) Reactive Groups of Labeling Reagents

[0014] The use of non-radioactive labels in biochemistry and molecularbiology has grown exponentially in recent years. Among the variouscompounds used as non-radioactive labels, aromatic dyes that producefluorescent or luminescent signal are especially useful. Notableexamples of such compounds include fluorescein, rhodamine, coumarin andcyanine dyes such as Cy3 and Cy5. Composite dyes have also beensynthesized by fusing two different dyes together (Lee et al., (1992)Nucl. Acids Res. 20; 2471-2488; Lee et al., U.S. Pat. No. 5,945,526 andWaggoner et al., in U.S. Pat. No. 6,008,373, all of which are herebyincorporated by reference).

[0015] Non-radioactive labeling methods were initially developed toattach signal-generating groups onto proteins. This was achieved bymodifying labels with chemical-groups such that they would be capable ofreacting with the amine, thiol, and hydroxyl groups that are naturallypresent on proteins. Examples of reactive groups that were used for thispurpose included activated esters such as N-hydroxysuccinimide esters,isothiocyanates and other compounds. Consequently, when it becamedesirable to label nucleotides and nucleic acids by non-radioactivemeans, methods were developed to convert nucleotides and polynucleotidesinto a form that made them functionally similar to proteins. Forinstance, U.S. Pat. No. 4,711,955 (incorporated by reference) disclosedthe addition of amines to the 8-position of a purine, the 5-position ofa pyrimidine and the 7-position of a deazapurine. The same methods thatcould add a label to the amine group of a protein could now be appliedtowards these modified nucleotides.

[0016] Among the compounds used as fluorescent labels, the cyanine-baseddyes have become widely used since they have high extinctioncoefficients and narrow emission bands. Furthermore, modifications canbe made in their structure that can alter the particular wavelengthswhere these compounds will absorb and fluoresce light. The cyanine dyeshave the general structure comprising two indolenine based ringsconnected by a series of conjugated double bonds. The dyes areclassified by the number (n) of central double bonds connecting the tworing structures; monocarbocyanine or trimethinecarbocyanine when n=1;dicarbocyanine or pentamethinecarbocyanine when n=2; and tricarbocyanineor heptamethinecarbocyanine when n=3. The spectral characteristics ofthe cyanine dyes have been observed to follow specific empirical rules.For example, each additional conjugated double bond between the ringswill raise the absorption and, emission maximum about 100 nm. Thus, whena compound with n=1 has a maximum absorption of approximately 550 nm,equivalent compounds with n=2 and n=3 will have maximum absorptions of650 nm and 750 nm respectively. Addition of aromatic groups to the sidesof the molecules can shift the absorption by 15 nm to a longerwavelength. The groups comprising the indolenine ring can alsocontribute to the absorption and emission characteristics. Using thevalues obtained with gem-dimethyl group as a reference point, oxygensubstituted in the ring for the gem-dimethyl group decreases theabsorption and emission maxima by approximately 50 nm. In contrast,substitution of sulfur increases the absorption and emission maxima byabout 25 nm. R groups on the aromatic rings such as alkyl,alkyl-sulfonate and alkyl-carboxylate have little effect on theabsorption and emission maxima of the cyanine dyes (U.S. Pat. No.6,110,630).

[0017] Cyanine dyes synthesized with arms containing functional groupshave been prepared with iodoacetamide, isothiocyanate and succinimidylesters that react with sulfhydryl groups on proteins (Ernst, etal.,(1989), Cytometry 10, 3-10; Mujumdar, et al., (1989), Cytometry 10,11-19; Southwick, et al., (1990) Cytometry 11, 4187-430). A new seriesof modified dyes were prepared which contained a sulfonate group on thephenyl portion of the indolenine ring. (Mujumdar et al., (1993)Bioconjugate Chemistry 4; 105-111 hereby incorporated by reference) thatincreased the water solubility of the dyes. These dyes were activated bytreatment with disuccinimidyl carbonate to form succinimidyl esters thatwere then used to label proteins by substitution at the amine groups.Other activating groups have since been placed on the cyanine dyes. InU.S. Pat. No. 5,627,027 and U.S. Pat. No. 5,268,486 (incorporated byreference), cyanine dyes were prepared which comprise isothiocyanate,isocyanate, monochlorotriazine, dichlorotriazine, mono or di-halogensubstituted pyridine, mono or di-halogen substituted diazine, aziridine,sulfonyl halide, acid halide, hydroxy-succinimide ester,hydroxy-sulfosuccinimide ester, imido esters, glyoxal groups andaldehydes and other groups, all of which can form a covalent bond withan amine, thiol or hydroxyl group on a target molecule.

[0018] In U.S. Pat. No. 6,110,6,30 (incorporated by reference), cyaninedyes were prepared with a series of reactive groups derived fromN-hydroxynaphthalimide. These groups included hydroxysuccinimide,para-nitrophenol, N-hydroxyphtalimide and N-hydroxynaphtalimide all ofwhich can react with nucleotides modified with primary amines. The samechemical reactions that have been described above have also been used inU.S. Pat. No. 6,114,350 (incorporated by reference) but with theconstituents reversed. In this disclosure, the cyanine dyes weremodified with amine, sulfhydryl or hydroxyl groups and the targetmolecules were modified to comprise the appropriate reactive groups.

[0019] Cyanine dyes containing arms that comprise reactive functionalgroups have been prepared by the general scheme in which the entireheterocyclic compound comprising the two indolenine structures and theintervening unsaturated chain was synthesized first; the terminalreactive groups or any other functionality necessary to link the dyes toproteins or nucleic acids were then added after the completion of thewhole dimeric dye unit.

[0020] (2) Linker Arms for Connecting Labels to Targets

[0021] Labeled nucleotides have been used for the synthesis of DNA andRNA probes in many enzymatic methods including terminal transferaselabeling, nick translation, random priming, reverse transcription, RNAtranscription and primer extension. Labeled phosphoramidite versions ofthese nucleotides have also been used with automated synthesizers toprepare labeled oligonucleotides. The resulting labeled probes arewidely used in such standard procedures as northern blotting, Southernblotting, in situ hybridization, RNAse protection assays, DNA sequencingreactions, DNA and RNA microarray analysis and chromosome painting.

[0022] There is an extensive literature on chemical modification ofnucleic acids by means of which a signal moiety is directly orindirectly attached to a nucleic acid. Primary concerns of this art havebeen with regard to which site in a nucleic acid is used for attachmenti.e. sugar, base or phosphate analogues and whether these sites aredisruptive or non-disruptive (see for instance the disclosures of U.S.Pat. No. 4,711,955 and U.S. Pat. No. 5,241,060; both patentsincorporated by reference) the chemistry at the site of attachment thatallows linkage to a reactive group or signaling moiety a spacer groupusually consisting of a single aromatic group (U.S. Pat. Nos. 4,952,685and 5,013,831, both hereby incorporated by reference) or a carbon/carbonaliphatic chain to provide distance between the nucleic acid and areactive group or signaling moiety and a reactive group at the end ofthe spacer such as an OH, NH, SH or some other group that can allowcoupling to a signaling moiety and the nature of the signaling moiety.

[0023] Although the foregoing have all been descriptions of the variousaspects that are concerned with the synthesis of modified nucleotidesand polynucleotides, they have also been shown to be significant factorswith regard to the properties of the resultant nucleotides andpolynucleotides. Indeed, there have been numerous demonstrations thatthe modified nucleotides described in the present art have shortcomingscompared to unmodified nucleotides.

[0024] For instance, these factors can have major impact on the abilityof these modified nucleotides to be incorporated by polymerases. Aconsequence of this is that when using a modified base as the solesource of that particular nucleotide, there may be a loss in the amountof nucleic acid synthesis compared to a reaction with unmodifiednucleotides. As a result of this, modified nucleotides are usuallyemployed as part of a mixture of modified and unmodified versions of agiven nucleotide. Although this restores synthesis to levels comparableto reactions without any modified nucleotides, a bias is often seenagainst the use of the modified version of the nucleotide. As such, thefinal proportion of modified/unmodified nucleotide may be much lowerthan the ratio of the reagents. Users then have a choice of either usingnucleic acids that are minimally labeled or of decreased yields. Whencomparable modified nucleotides are used that only comprise a linker armattached to a base (such as allylamine dUTP) difficulties withincorporation are seldom seen. As such, the foregoing problem is likelyto be due to the interactions of the label with either the polymerase orthe active site where synthesis is taking place.

[0025] Difficulties in the use of polymerases can be bypassed by the useof oligonucleotide synthesizers where an ordered chemical joining ofphosphoramidite derivatives of nucleotides can be used to producelabeled nucleic acids of interest. However, the presence of signalagents on modified nucleotides can even be problematic in this system.For instance, a phosphoramidite of a modified nucleotide may display aloss of coupling efficiency as the chain is extended. Although this maybe problematic in itself, multiple and especially successive use ofmodified nucleotides in a sequence for a synthetic oligonucleotide canresult in a drastic cumulative loss of product. Additionally, chemicalsynthesis is in itself not always an appropriate solution. There may becircumstances where labeled nucleic acids need to be of larger lengthsthan is practical for a synthesizer. Also, an intrinsic part ofsynthetic approaches is a necessity for a discrete sequence for thenucleic acid. For many purposes, a pool or library of nucleic acidswould require an impractically large number of different species forsynthetic approaches.

[0026] An example of a method to increase the yield of labeledoligonucleotides or polynucleotide is to use a non-interfering groupsuch as an allylamine modified analogue during synthesis by either apolymerase or an oligonucleotide synthesizer. Labeling is then carriedout post-synthetically by attachment of the desired group through thechemically reactive allylamine moieties. However, in this case, althoughincorporation or coupling efficiency may be restored, there may still beproblems of the coupling efficiencies of attachment of the desired groupto the allylamine. For instance, coupling of labels to allylaminemoieties in a nucleic acid is dramatically less efficient fordouble-stranded DNA compared to single-stranded targets. In addition topotential yield problems, the functionality of the modification may beaffected by how it is attached to a base. For instance if a hapten isattached to a base, the nature of the arm separating the hapten from thebase may affect its accessibility to a potential binding partner. When asignal generating moiety is attached through a base, the nature of thearm may also affect interactions between the signal generating moietyand the nucleotide and polynucleotide.

[0027] Attempts to limit these deleterious interactions have beencarried out in several ways. For instance, attachment of the arm to thebase has been carried out with either a double bond alkene group (U.S.Pat. No. 4,711,955) or a triple bond alkyne group (U.S. Pat. No.5,047,519) thereby inducing a directionality of the linker away from thenucleotide or polynucleotide. However, this approach is of limitedutility since this rigidity is limited to only the vicinity of theattachment of the linker to the base. In addition, attempts at limitinginteractions have been carried out by having the arm displace the activeor signal group away from the nucleotide or polynucleotide bylengthening the spacer group. For instance, a commercially availablemodified nucleotide included a seven carbon aliphatic chain (Cat. No.42724, ENZO Biochem, Inc. New York, N.Y.) between the base and a biotinmoiety used for signal generation. This product was further improved bythe substitution of linkers with 11 or even 16. carbon lengths (Cat.Nos. 42722 and 42723, also available from ENZO Biochem, Inc. New York,N.Y.). A comparison was also carried out using different length linkerarms and a cyanine dye labeled nucleotide (Zhu et al., 1994 Nucl. AcidRes. 22; 3418-3422). A direct improvement in efficiency was noted as thelength was increased from 10 to 17 and from 17 to 24. However, even withthe longest linker, it could be seen that there was incompletecompensation for the presence of the fluorescent marker in terms ofefficiency. This may be a result of the fact that due to the flexibilityof the aliphatic carbon chain used for this spacer segment, the reportergroups will seldom be found in a conformation where they are completelyextended away from the nucleotide itself. Thus, although this approachchanged the length of the linker, it was not a change in the flexiblenature of the spacer.

[0028] In an attempt to circumvent this problem, in U.S. Pat. No.5,948,648, Khan et al. have disclosed the use of multiple alkyne oraromatic groups connecting a marker to a nucleotide. However, thismethod employs highly non-polar groups in the linker that may induceinteraction between the linker and the marker, thereby limiting itseffectiveness by decreasing coupling efficiencies or by increasingnon-specific binding by labeled compounds that include these groups. Inaddition, these groups may decrease the water solubility of either theelabeled compound or various intermediates used to make the labeledcompound.

[0029] The continued difficulties in using activated or labelednucleotides which have incorporated the foregoing features demonstratesthat there are still deleterious interactions occurring between thebase, oligonucleotide or polynucleotide and the moiety at the end of thearm in methods of the previous art. Although the foregoing has beendescribed with respect to attachment to nucleic acids, these problemsare shared with other groups for which it may be useful to attach amarker or label.

[0030] (3) Porphyrin Fluorescent Dyes as Labels

[0031] Assays that employ fluorescently labeled probes depend uponillumination at one particular wavelength and detection of the emissionat another wavelength (the Stokes shift). There exists an extensiveliterature on the variety of compounds that have variousexcitation/emission spectral characteristics suitable for such assays.When fluorescent compounds are used for comparative expression analysis,the ability to carry out signal detection simultaneously for each labeldepends upon how marked is the difference between the labels. Thus,fluorophores such as Cy 3 and Cy 5 are commonly used in expressionanalysis since they have emission peaks at 570 and 667 respectively. Oneclass of compounds that has not been effectively exploited for thisanalysis are the porphyrins.

[0032] The ability of porphyrins to absorb light energy and efficientlyrelease it has been used in a number of other systems. For example,light induced cleavage of nucleic acids can be carried out by a numberof metallo-porphyrins that are either free in solution or attached to asequence specific oligonucleotide (Doan et al., (1986) Biochemistry 26;6736-6739). One application of this system has been the targeting andkilling of cancer cells through light induced DNA damage afterabsorption of metallo-phorphyrins (Moan et al., (1986) Photochemistryand Photobiology 43; 681-690). Another example of the high energeticability of metallo-phorphyrins can be seen with their use as catalyticagents (Forgione et al., U.S. Pat. No. 4,375,972) for non-enzymaticchemiluminescence. Futhermore, there are cases where phorphyrins havebeen used as labeling reagents, for example Roelant et al in U.S. Pat.No. 6,001,573 and Hendrix in U.S. Pat. No. 5,464,741 (herebyincorporated by reference) where Pd octaethylporphyrins were convertedto the isothiocyanate and used as labeling reagents particularly for usein immunoassays. However, in these cases metallic phorphyrins wereexclusively used.

[0033] The drawback of the use of metallo-phorphyrins is that thedestructive abilities of these compounds are counter-productive whenused in array analysis or other assay systems which require themaintenance of the integrity of the nucleic acid strands of analytes orprobes. Therefore, it would be highly advantageous to be able to utilizeporphyrins for their fluorescent and chemiluminescent properties whileeliminating their nucleic acid destructive properties.

[0034] (4) Alterations in Fluorescent Properties

[0035] In previous art, it has been shown that the addition ofphenylacetylene groups to anthracene increases the emission maxima 72nm. (Maulding and Roberts, 1968 J Org Chem). Furthermore, the Stokesshift, the difference between the absorption and emission maxima, wasalso increased by the addition of the phenyl acetylene group to theanthracene dye. Specifically the difference of 6 nm was increased to 31nm following the addition of two phenyl acetylene groups. When thephenyl acetylene group was added to naphthacene the difference betweenthe absorption and emission maxima increased from 7 nm to 32 nm.Furthermore, the quantum yields of anthracene and naphtacene wassignificantly increased by the addition of the phenyl acetylene groupsto them.

[0036] The application of this effect was limited to these compoundsbecause the chemistries and reactions used for the addition of thesesubstituents required ketone or aldehyde groups. Also, addition ofunsaturated groups to dyes has the undesired effect of potentiallydecreasing their solubility in aqueous solutions. In addition, themodified anthracene dyes described by Maulding and Roberts lacked anyreactive groups that could be used for attachment.

[0037] (5) Fluorescent Intercalators

[0038] Intercalating dyes have been used for the detection andvisualization of DNA in many techniques including the detection of DNAin electrophoresis gels, in situ hybridization, flow cytometry and realtime detection of amplification. An intercalating dye with a longhistory of popular usage is ethidium bromide. Ethidium bromide has theuseful properties of high affinity for nucleic acids and an increasedfluorescence after binding. This enhancement of fluorescence takes placewith both single-stranded and double-stranded nucleic acids with thedouble-stranded DNA showing a much more marked effect, generally aroundthirty-fold. Other dyes which exhibit increased fluorescence signal uponbinding to nucleic acid have been developed in recent years includingsuch compounds as acridine orange, SYBR Green and Picogreen. There iscontinually a need, however, for increased signal generation after thebinding or intercalation with nucleic acids especially for the use intechniques, such as real time amplification.

[0039] (6) Chemiluminescence

[0040] The use of chemiluminescent reagents for signal detection hasgained wider use in recent years. There are several different classes ofcompounds that can produce luminescent signals including-1,2-dioxetanesand luminols. 1,2-Dioxetanes are four-membered rings which contain twoadjacent oxygens. Some forms of these compounds are very unstable andemit light as they decompose. On the other hand, the presence of anadamantyl group can lead to a highly stable form with a half-life ofseveral years (Wieringa et al. (1972) Tetrahedron Letters 169-172incorporated by reference). Use can be made of this property by using astable form of a 1,2-dioxetane as a substrate in an enzyme linked assaywhere the presence of the enzyme will transform the substrate into anunstable form thereby using chemiluminescence for signal generation.Enzymatic induction of a chemiluminescent signal has been describedwhere an adamantyl dioxetane derivative was synthesized with anadditional group that was a substrate for enzymatic cleavage (U.S. Pat.No. 5,707,559, Schaap et al. (1987) Tetrahedron Letters, 28; 935-938;Schaap et al. (1987) Tetrahedron Letters, 28; 1159-1163, all of whichare incorporated by reference). In the presence of the appropriateenzyme, cleavage would take place and an unstable compound would beformed that emitted light as it decomposes.

[0041] A common design of dioxetane derivatives for this method isattachment of an aryl group that has hydroxyl substituents which containprotecting groups. The removal of the protecting group by theappropriate enzyme results in a negatively charged oxygen. Thisintermediate is unstable and leads to the decomposition of the compoundand the emission of light. Various 1,2-dioxetane derivatives have beendeveloped that can be activated by different enzymes depending upon thenature of the protecting group. Enzymes that have been described aspotentially useful for this purpose have included alkaline phosphatase,galactosidase, glucosidase, esterase, trypsin, lipase, and phospholipaseamong others (for instance, see U.S. Pat. No. 4,978,614, incorporatedherein by reference).

[0042] Variations of this basic method have also been disclosed. Forexample, Urdea has disclosed (U.S. Pat. No. 5,132,204, incorporated byreference) stable 1,2-dioxetanes derivatives which require the activityof two enzymes in order to produce a signal. Haces has disclosed amethod where the decomposition of the 1,2-dioxetane is triggered by anenzymatic or chemical reaction which releases a terminal nucleophile(U.S. Pat. No. 5,248,618 incorporated by reference). This can nowundergo an intramolecular substitution reaction, thereby liberating aphenoxy group which triggers the decomposition of the 1,2-dioxetane. Thechain where the intramolecular reaction takes place is made up of singlebonds thus allowing complete rotational freedom around all the bonds andrelying on a random interaction between the groups participating in theintramolecular reaction.

[0043] Despite improvements within the field of chemiluminescentsignaling there still exists the need for new substrates and reagents.Many of the substrates that are currently available produce a high levelof background due to enzyme independent triggering of the decompositionof the substrate and release of chemiluminescent signal. Therefore, anew type of 1,2-dioxetane which is more stable in the absence of anenzyme would be a desirable reagent.

[0044] (7) Real Time Detection Through Fluorescence

[0045] Amplification of nucleic acids from clinical samples has become awidely used technique. The first methodology for this process, thePolymerase Chain Reaction (PCR), was described by Mullis et al. in U.S.Pat. No. 4,683,202 hereby incorporated by reference. Since that time,other methodologies such as Ligation Chain Reaction (LCR) (U.S. Pat. No.5,494,810), GAP-LCR (U.S. Pat. No. 6,004,286), Nucleic Acid SequenceBased Amplification (NASBA) (U.S. Pat. No. 5,130,238), StrandDisplacement Amplification (SDA) (U.S. Pat. No. 5,270,184 and U.S. Pat.No. 5,455,166) and Loop Mediated Amplification (U.S. patent applicationSer. No. 09/104,067; European Patent Application Publication No. EP 0971 039 A) have been described, all of which are incorporated byreference. Detection of an amplified product derived from theappropriate target has been carried out in number of ways. In theinitial method described by Mullis et al., gel analysis was used todetect the presence of a discrete nucleic acid species. Identificationof this species as being indicative of the presence of the intendedtarget was determined by size assessment and the use of negativecontrols lacking the target sequence. The placement of the primers usedfor amplification dictated a specific size for the product fromappropriate target sequence. Spurious amplification products made fromnon-target sequences were unlikely to have the same size product as thetarget derived sequence. Alternatively, more elaborate methods have beenused to examine the particular nature of the sequences that are presentin the amplification product. For instance, restriction enzyme digestionhas been used to determine the presence, absence or spatial location ofspecific sequences. The presence of the appropriate sequences has alsobeen established by hybridization experiments. In this method, theamplification product can be used as either the target or as a probe.

[0046] The foregoing detection methods have historically been used afterthe amplification reaction was completed. More recently, methods havebeen described for measuring the extent of synthesis during the courseof amplification, i.e. “real-time” detection. For instance, in thesimplest system, an intercalating agent is present during theamplification reaction (Higuchi in U.S. Pat. No. 5,994,056 and Wittweret al., U.S. Pat. No. 6,174,670; both of which are hereby incorporatedby reference). This method takes advantage of an enhancement offluorescence exhibited by the binding of an intercalator todouble-stranded nucleic acids. Measurement of the amount of fluorescencecan take place post-synthetically in a fluorometer after the reaction isover, or real time measurements can be carried out during the course ofthe reaction by using a special PCR cycler machine that is equipped witha fluorescence detection system and uses capillary tubes for thereactions (U.S. Pat. No. 5,455,175 and U.S. Pat. No. 6,174,670 herebyincorporated by reference). As the amount of double-stranded materialrises during the course of amplification, the amount of signal alsoincreases. The sensitivity of this system depends upon a sufficientamount of double-stranded nucleic acid being produced that generates asignal that is distinguishable from the fluorescence of a) unboundintercalator and b) intercalator molecules bound to single-strandedprimers in the reaction mix. Specificity is derived from the nature ofthe amplification reaction itself or by looking at a Tm profile of thereaction products. Although the initial work was done with EthidiumBromide, SYBR Green™ is more commonly used at the present time. Avariation of this system has been described by Singer and Haugland inU.S. Pat. No. 6,323,337 B1 (incorporated by reference), where theprimers used in PCR reactions were modified with quenchers therebyreducing signal generation of a fluorecent intercalator that was boundto a primer dimer molecule. Signal generation from target derivedamplicons could still take place since amplicons derived from targetsequences comprised intercalators bound to segments that weresufficiently distant from the quenchers.

[0047] Another method of analysis that depends upon incorporation hasbeen described by Nazarenko (U.S. Pat. No. 5,866,336; incorporated byreference). In this system, signal generation is dependent upon theincorporation of primers into double-stranded amplification products.The primers are designed such that they have extra sequences added ontotheir 5′ ends. In the absence of amplification, stem-loop structures areformed through intramolecular hybridization that consequently bring aquencher into proximity with an energy donor thereby preventingfluorescence. However, when a primer becomes incorporated intodouble-stranded amplicons, the quencher and donor become physicallyseparated and the donor is now able to produce a fluorescent signal. Thespecificity of this system depends upon the specificity of theamplification reaction itself. Since the stem-loop sequences are derivedfrom extra sequences, the Tm profile of signal generation is the samewhether the amplicons were derived from the appropriate target moleculesor from non-target sequences.

[0048] In addition to incorporation based assays, probe based systemshave also been used for real-time analysis. For instance, a dual probesystem can be used in a homogeneous assay to detect the presence ofappropriate target sequences. In this method, one probe comprises anenergy donor and the other probe comprises an energy acceptor (EuropeanPatent Application Publication No. 0 070 685 by Michael Heller,published Jan. 26, 1983). Thus, when the target sequence is present, thetwo probes can bind to adjacent sequences and allow energy transfer totake place. In the absence of target sequences, the probes remainunbound and no energy transfer takes place. Even if by chance, there arenon-target sequences in a sample that are sufficiently homologous thatbinding of one or both probes takes place, no signal is generated sinceenergy transfer would require that both probes bind and that they be ina particular proximity to each other. Advantage of this system has beentaken by Wittwer et al., in U.S. Pat. No. 6,174,670 (incorporated byreference) for real time detection of PCR amplification using thecapillary tube equipped PCR machine described previously. The primerannealing step during each individual cycle can also allow thesimultaneous binding of each probe to target sequences providing anassessment of the presence and amount of the target sequences. In afurther refinement of this method, one of the primers comprises anenergy transfer element and a single energy transfer probe is used.Labeled probes have also been used in conjunction with fluorescentintercalators to allow the specificity of the probe methodology to becombined with the enhancement of fluorescence derived from binding tonucleic acids. This was first described in U.S. Pat. No. 4,868,103 andlater applied to amplification reactions in PCT Int. Appl. WO 99/28500(both documents incorporated by reference).

[0049] Probes have also been used that comprise an energy donor and anenergy acceptor in the same nucleic acid. In these assays, the energyacceptor “quenches” fluorescent energy emission in the absence ofappropriate complementary targets. In one system described by Lizardi etal. in U.S. Pat. No. 5,118,801, “molecular beacons” are used where theenergy donor and the quencher are kept in proximity by secondarystructures with internal base pairing. When the target sequences arepresent, complementary sequences in the Molecular Beacons allowhybridization events that destroy this secondary structure therebyallowing energy emission. In another system that has been termed Taqman,use is made of the double-stranded selectivity of the exonucleaseactivity of Taq polymerase (Gelfand et al., U.S. Pat. No. 5,210,015).When target molecules are present, hybridization of the probe tocomplementary sequences converts the single-stranded probe into asubstrate for the exonuclease. Degradation of the probe separates thedonor from the quencher thereby releasing light.

[0050] (8) Primer Binding Sequences in Analytes

[0051] One of the characteristics of eucaryotic mRNA is the presence ofpoly A tails at their 3′ ends. This particular feature has provided amajor advantage in working with mRNA since the poly A segment can beused as a universal primer binding site for synthesis of cDNA copies ofany eucaryotic mRNA. However, this has also led to a certain bias in RNAstudies, since the 3′ ends of mRNA are easily obtained and thoroughlystudied but the 5′ ends lack such consensus sequences. Thus, a largenumber of systems have been described whose major purpose has been togenerate clones that have complete representation of the original 5′ endsequences. This has also been carried over in array analysis forcomparative transcription studies. Since substantially all systems usedfor this purpose are initiated by oligo T priming at the 3′ end of mRNA,sequences downstream are dependent upon the continuation of synthesisaway from the 3′ starting point. However, it is well known that there isan attenuation effect of polymerization as polymerases frequently falloff of templates after synthesis of a particular number of bases.Another effect is generated by the presence of RNase H that is acomponent of most reverse transcriptases. Paused DNA strands may allowdigestion of the RNA near the 3′ end of the DNA thereby separating theuncopied portion of the RNA template from the growing DNA strand. Thiseffect may also occur randomly during the course of cDNA synthesis. Assuch, representation of sequences is inversely proportional to theirdistance from the 3′ poly A primer site.

[0052] Although prior art has capitalized extensively on poly A segmentsof RNA, it should be recognized that poly A mRNA represents only aportion of nucleic acids in biological systems. Another constraint inprior art is that the use of poly A tails is only available ineucaryotic mRNA. Two areas of especial interest are unable to enjoy thisbenefit. One area is bacterial mRNA since they intrinsically lack poly Aadditions. The second are is heterologous RNA in eucaryotic systems. Forany particular eucaryotic gene, there is a considerable amount ofgenetic information that is present in heterologous RNA that is lost bythe use of polyadenylated mature forms of transcripts that comprise onlyexon information.

[0053] The lack of primer consensus sequence in these systems hasnecessitated the use of alternatives to oligo T priming. In prior art,bacterial expression studies have been carried out by random primingwith octamers (Sellinger et al., 2000 Nature Biotechnology 18;1262-1268), a selected set of 37 7-mers and 8-mers (Talaat et al., 2000Nature Biotechnology 18; 679-682) and a set of 4,290 gene specificprimers (Tao et al., 1999 J. Bact. 181; 6425-6490). The use of largesets of primers as represented by random primers and set of genespecific primers requires high amounts of primers to drive the reactionand should exhibit poor kinetics due to the sequence complexity of theprimers and targets. I.e. for any given sequence in an analyte, there isonly a very minute portion of the primers that are complementary to thatsequence. Large sets of random primers also have the capacity to useeach other as primers and templates thereby generating nonsense nucleicacids and decreasing the effective amounts of primers available.Attempts to improve the kinetics of priming by increasing the amounts ofrandom oligonucleotides is very limited. First off, there are physicalconstraints in the amount of oligonucleotides that are soluble in areaction mixture. Secondly, increases in the amount of primers isself-limiting since increased primer concentrations results in increasedself-priming, thereby generating more nonsense sequences and absorptionof reagents that would otherwise be used for analyte dependentsynthesis. Lower concentrations can theoretically be used by decreasingthe complexity (i.e. sequence length) of the primers, but restraints arethen imposed upon the stability of hybrid formation. On the other hand,the discrete sub-set of 7-mers and 8-mers described above requiresknowledge of the complete genome of the intended target organism. Assuch, these will only be used with completely sequenced organisms, and aunique set has to be individually developed for each target organismthus limiting its application. Consensus sequences can be enzymaticallyadded by RNA ligation or poly A polymerase but both of these are slowinefficient processes. Thus there exists a need for methods andcompositions that can efficiently provide stable priming of a largenumber of non-polyadenylated templates of variable or even unknownsequence while maintaining a low level of complexity.

[0054] Methods have also been described for the introduction ofsequences into analytes for the purpose of amplification. For instance,oligonucleotides have also been described that comprise a segmentcomplementary to a target sequence and a segment comprising a promotersequence where the target is either a selected discrete sequence or anatural poly A sequence (U.S. Pat. No. 5,554,516 and U.S. Pat. No.6,338,954 (both patents incorporated by reference)). After hybridizationto a target mRNA, RNAse H is used to cleave a segment of the analytehybridized to the complementary segment and then extend the 3′ end ofthe analyte using the promoter segment as a template. Since theoligonucjetotide that is used for these methods has a homogeneousnature, this particular method relies upon the extension reaction beinginitiated before the endonuclease reaction completes digestion of thecomplementary segment of the analyte.

SUMMARY OF THE INVENTION

[0055] The present invention provides a labeling reagent for labeling atarget, the labeling reagent comprising a marker moiety M and a reactivegroup R

M-R

[0056] wherein the marker moiety M and the reactive group R arecovalently linked together, the M comprising at least one moiety thatcomprises a ligand, a dye, or both a ligand and a dye; and the reactivegroup R being capable of forming a carbon-carbon linkage with thetarget.

[0057] The present invention also provides a process for labeling atarget, the process comprising the steps of (a) providing: (i) thetarget; (ii) a labeling reagent comprising a marker moiety M and areactive group R

M-R

[0058] wherein the marker moiety M and the reactive group R arecovalently linked together, the M comprising at least one moiety thatcomprises a ligand, a dye, or both a ligand and a dye; and the reactivegroup R being capable of forming a carbon-carbon linkage with thetarget; and (b) reacting the target (i) and the labeling reagent (ii)under conditions such that a carbon-carbon linkage forms between thetarget (i) and the labeling reagent (ii), thereby labeling the target(i) with the marker moiety M.

[0059] This invention also provides a labeled target, the target havingbeen labeled by a process comprising the steps of (a) providing: (i) thetarget; (ii) a labeling reagent comprising a marker moiety M and areactive group R

M-R

[0060] wherein the marker moiety M and the reactive group R arecovalently linked together, the M comprising at least one moiety thatcomprises a ligand, a dye, or both a ligand and a dye; and the reactivegroup R being capable of forming a carbon-carbon linkage with thetarget; (b) reacting the target (i) and the labeling reagent (ii) underconditions such that a carbon-carbon linkage forms between the target(i) and the labeling reagent (ii), thereby labeling the target (i) withthe marker moiety M.

[0061] Also provided by this invention is a process for preparing acyanine dye labeling reagent, the process comprising the steps of (a)providing:

[0062] (i) a first intermediate compound comprising:

[0063] wherein X₁ comprises carbon, oxygen, nitrogen or sulfur, and

[0064] (ii) a second intermediate compound comprising:

[0065] wherein X₁ comprises carbon, oxygen, nitrogen or sulfur; whereinat least one of R₁ through R₁₀ comprises a reactive group capable offorming a carbon-carbon linkage with a target, and (ii) linking reagentssuitable for linking the first intermediate compound and the secondintermediate compound; (b) forming a reaction mixture comprising thefirst intermediate compound (i), the second intermediate compound (ii),and the linking reagents under conditions to link (i) and (ii) to form

[0066] wherein at least one of R₁ through R₁₀ comprises a reactive groupcapable of forming a carbon-carbon linkage with a target, and wherein nis an integer of 1, 2 or 3, and wherein X₁ and X₂ independently comprisecarbon, oxygen, nitrogen or sulfur.

[0067] Further provided by this invention is a labeling reagentcomprising an aphenylic analog of a rhodamine dye, the analog comprisingat least one reactive group for attaching the labeling reagent to atarget, the at least one reactive group being attached directly to theanalog or indirectly through a linker arm.

[0068] The present invention also concerns a labeled nucleotidecomprising an aphenylic analog of a rhodamine dye, wherein the dye isattached directly to the nucleotide or indirectly through a linker.

[0069] This invention also provides a labeled target comprising

T—L—M

[0070] wherein T is a target, M is a marker moiety and L is a chemicalgroup covalently linking the M to T, the chemical group L comprising abackbone that comprises at least one rigid group that comprises one ormore of:

[0071] (d) multimers of (a), (b) or (c), and (e) any combinations of(a), (b), (c) and (d).

[0072] The present invention also provides a labeling reagent comprising

R—L—M

[0073] wherein R is a reactive group, M is a marker moiety and L is achemical group covalently linking the M to R, the chemical group Lcomprising a backbone that comprises at least one rigid group thatcomprises one or more of:

[0074] (d) multimers of (a), (b) or (c), and (e) any combinations of(a), (b), (c) and (d).

[0075] Also provided is a labeled target comprising

T—L—M

[0076] wherein T is a target, M is a marker moiety and L is a chemicalgroup covalently linking the M to T, the chemical group L comprising abackbone that comprises at least two consecutive polar rigid units.

[0077] Additionally provided is a labeling reagent comprising

R—L—M

[0078] wherein R is a reactive group, M is a marker moiety and L is achemical group covalently linking the M to R, the chemical group Lcomprising a backbone that comprises at least two consecutive polarrigid units.

[0079] The invention herein also provides a labeled target comprising

T—L—M

[0080] wherein T is a target, M is a marker moiety and L is a chemicalgroup covalently linking the M to T, the chemical group L comprising abackbone that comprises at least two consecutive peptide bonds.

[0081] Another aspect of this invention is a labeling reagent comprising

R—L—M

[0082] wherein R is a reactive group, M is a marker moiety and L is achemical group covalently linking the M to R, the chemical group Lcomprising a backbone that comprises at least two consecutive peptidebonds.

[0083] Another aspect concerns a labeling reagent comprising anonmetallic porphyrin, the reagent comprising:

[0084] wherein R₀ is a reactive group and is attached directly orindirectly to the nonmetallic porphyrin, and R₁ through R₈ independentlycomprise hydrogen, aliphatic, unsaturated aliphatic, cyclic,heterocyclic, aromatic, heteroaromatic, charged or polar groups, or anycombinations of the foregoing.

[0085] Further described and provided is a labeled target comprising anonmetallic porphyrin, the reagent comprising:

[0086] wherein T is a target molecule attached directly or indirectly tothe nonmetallic porphyrin and R₁ through R₈ independently comprisehydrogen, aliphatic, unsaturated aliphatic, cyclic, heterocyclic,aromatic, heteroaromatic, charged or polar groups, or any combinationsof the foregoing.

[0087] Another part of the present invention is a process fordetermining the amount of a nucleic acid in a sample of interest, theprocess comprising the steps of: (a) providing: (i) the sample ofinterest; (ii) a dye comprising a first phenanthridinium moiety linkedto a second phenanthridinium moiety through the phenyl group in each ofthe first and second phenanthridinium moieties; (iii) reagents forcarrying out dye binding, hybridization, strand extension, or anycombination thereof; (b) forming a mixture of (i), (ii) and (iii) above,to produce a complex comprising the dye (ii) and any nucleic acid thatmay be present in the sample of interest (i); (c) illuminating themixture formed in step (b) at a wavelength below 400 nanometers (nm);and (d) measuring fluorescent emission from the illuminated mixture instep (c), the emission being proportional to the quantity of any nucleicacid present in the sample of interest (i).

[0088] The present invention also provides a composition comprising atleast one of the following dye structures:

[0089] Also provided by the present invention is the use of thejust-described compositions in a process for determining the amount of anucleic acid in a sample of interest, the process comprising the stepsof: (a) providing: (i) the sample of interest; (ii) the dye (a), (b),(c) or (d) from the composition just described; (iii) reagents forcarrying out dye binding, hybridization, strand extension, or anycombination thereof; (b) forming a mixture of (i), (ii) and (iii) above,to produce a complex comprising the dye (ii) and any nucleic acid thatmay be present in the sample of interest (i); (c) illuminating themixture formed in step (b) at a first wavelength; and (d) measuring at asecond wavelength any fluorescent emission from the illuminated mixturein step (c), the emission being proportional to the quantity of anynucleic acid present in the sample of interest (i).

[0090] This invention provides a heterodimeric dye composition, thecomposition comprising a first dye that comprises a phenanthridiniummoiety; and a second dye that is different from the first dye, thesecond dye being attached through the phenyl ring of the phenanthridiummoiety.

[0091] This invention also provides a process for determining the amountof a nucleic acid in a sample of interest using the last-describedcomposition. The process comprises the steps of: (a) providing: (i) thesample of interest; (ii) the dye last-described; (iii) reagents forcarrying out dye binding, hybridization, strand extension, or anycombination thereof; (b) forming a mixture of (i), (ii) and (iii) above,to produce a complex comprising the dye (ii) and any nucleic acid thatmay be present in the sample of interest (i); (c) illuminating themixture formed in step (b) at a first wavelength; and (d) measuring at asecond wavelength any fluorescent emission from the illuminated mixturein step (c), the emission being proportional to the quantity of anynucleic acid present in the sample of interest (i).

[0092] A chemiluminescent reagent is also provided by this invention,the chemiluminescent reagent having the structure:

[0093] wherein Q comprises a cycloalkyl or polycycloalkyl group attachedcovalently to the 4-membered ring portion of the dioxetane abovedirectly or indirectly through a linkage group; wherein Z compriseshydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl or cycloheteroalkyl; and wherein R₁ and R₂ comprise chemicalmoieties attached to different sites of a cyclic ring attached to thedioxetane, and wherein R₁ is enzymatically converted into R₁* whichcomprises a chemical reactive group G₁, and wherein R₂ is attached tothe cyclic ring through an oxygen, nitrogen or sulfur atom and comprisesa chemical reactive group G₂ which reacts with the G₁ to convert thedioxetane to an unstable light-emitting dioxetane form.

[0094] Using the last-described composition, the invention furtherprovides a process for detecting the presence or quantity of enzymaticactivity of interest in a sample. The process comprises the steps of:(a) providing: (i) the sample suspected of containing enzymaticactivity; (ii) the chemiluminescent reagent last-described; (ii)reagents and buffers for carrying out chemiluminescent reactions; (b)forming a mixture of: (1) (i), (ii) and (iii); or (2) (ii) and (iii) andcontacting the mixture of (ii) and (iii) with (i); (c) enzymaticallyconverting the chemiluminescent reagent just described (ii) into anunstable light-emitting dioxetane form; and (d) measuring the quantityof light generated by the enzymatic conversion in step (c).

[0095] Another chemiluminescent reagent provided by the presentinvention is one having the structure:

[0096] wherein Q comprises a cycloalkyl or polycycloalkyl group attachedcovalently to the 4-membered ring portion of the dioxetane abovedirectly or indirectly through a linkage group; wherein Z compriseshydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl or cycloheteroalkyl; and wherein R comprises a chemicallinker having a reactive site attached to the aromatic ring in thestructure; and wherein R¹ comprises a substrate for an non-cleavingenzymatic process, wherein the product of the enzymatic process leads tofurther chemical rearrangements that generate an unstable light emittingdioxetane form.

[0097] The invention also provides a process for detecting the presenceor quantity of enzymatic activity of interest in a sample using thelast-described chemiluminescent reagent. The process comprises the stepsof: (a) providing: (i) the sample suspected of containing enzymaticactivity; (ii) the chemiluminescent reagent last-described; (ii)reagents and buffers for carrying out chemiluminescent reactions; (b)forming a mixture of: (1) (i), (ii) and (iii); or (2) (ii) and (iii) andcontacting the mixture of (ii) and (iii) with (i); (c) enzymaticallyconverting the chemiluminescent reagent just described above (ii) intoan unstable light-emitting dioxetane form; and (d) measuring thequantity of light generated by the enzymatic conversion in step (c).

[0098] A dye composition is also provided by this invention, the dyecomposition having the formula

R-Fluorescent Dye

[0099] wherein R is covalently linked to the Fluorescent Dye comprisestwo or more members in combination from a) unsaturated aliphatic groups;b) unsaturated heterocyclic groups; c) aromatic groups; and wherein R iscapable of providing a conjugated system or an electron delocalizedsystem with the fluorescent dye.

[0100] A labeled target is further provided, the labeled target havingthe structure

[R-Dye]—target

[0101] wherein the Dye is a fluorescent dye, wherein R is covalentlylinked to the Dye, and wherein R comprises two or more members incombination from a) unsaturated aliphatic groups; b) unsaturatedheterocyclic groups; c) aromatic groups; and wherein R is capable ofproviding a conjugated system or an electron delocalized system with theDye.

[0102] Other embodiments and aspects of the present invention arefurther described below.

BRIEF DESCRIPTION OF THE FIGURES

[0103]FIG. 1 illustrates several examples of linker arms made with rigidpolar units.

[0104]FIG. 2 shows structures of various homodimers of Ethidium Bromide:

[0105] A) meta-EthD B) EthD-1 and C) EthD-2.

[0106]FIG. 3 illustrates the use of primers with first and second energytransfer elements: double stranded nucleic acid made from extension oftwo primers

[0107] double stranded SDA amplicon

[0108] Nested PCR

[0109] Ligase Chain Reaction.

[0110]FIG. 4 depicts variations of placement of primers in a doublestranded nucleic acid.

[0111]FIG. 5 is an illustration of the use of nucleotides with energytransfer elements.

[0112]FIG. 6 shows the use of matrix with energy transfer elements.

[0113]FIG. 7 is the spectrum of aphenylic analog of TAMRA.

[0114]FIG. 8 is the spectrum of aphenylic analog of Texas Red.

[0115]FIG. 9 is an outline of method for synthesizing a homodimer.

[0116]FIG. 10 depicts the results of illuminating meta-EtBr at 493 nm inthe presence and absence of DNA.

[0117]FIG. 11 shows the results of Illuminating meta-EtBr at 350 nm inthe presence and absence of DNA.

[0118]FIG. 12 shows the sequence of an HIV antisense amplicon andsequences of two primers and one probe used in the examples below toillustrate the novel use of energy transfer in the present invention.

[0119]FIG. 13 shows the use of a CNAC to eliminate a portion of a poly Atail followed by incorporation of an oligo C primer binding sequence.

[0120]FIG. 14 shows various steps for the synthesis of a dioxetanederivative.

[0121]FIG. 15 shows the enzymatic production of an unstable lightemitting form of a dioxetane.

DETAILED DESCRIPTION OF THE INVENTION

[0122] The present invention discloses novel methods and compositionsfor the preparation of compounds labeled with ligands and dyes. Includedwithin the present disclosure are novel labeling reagents, novel dyesand novel methods that can be used to synthesize the novel reagents ofthe present invention. The novel methods of the present invention mayalso be applied to the synthesis of compounds that have been describedpreviously.

[0123] 1. Labeling Reagents Which Participate in Carbon-Carbon BondFormation

[0124] One aspect of the present invention discloses novel labelingreagents that comprise a reactive group capable of creating acarbon-carbon bond between a marker or label and a desirable targetmolecule. This is in contrast to labeling reagents described in priorart which employed protein derived chemistries involving formation of abond between an amine, sulfhydryl or hydroxyl group and an appropriatereactive group. The novel labeling reagent of the present inventionshould provide a highly efficient means of attaching signal moieties todesirable target molecules. Thus, the novel labeling reagents of thepresent invention comprise a ligand or dye portion and a reactive groupcapable of creating a carbon-carbon bond. In addition, it may bedesirable to insert a linker arm that separates the ligand or dyeportion from the reactive group. This may provide more efficientcoupling between the novel labeling reagent and an intended targetmolecule. The presence and nature of the linker arm may also increasethe biological or chemical activity of the labeled target molecule. Thenovel reagents of the present invention can be used to label any targetmolecule that is capable of participating in bond formation with thereactive group of the labeling reagent. The target molecule may be inits native state or it may have been modified to participate information of a carbon-carbon bond with the novel labeling reagent.

[0125] Ligands that may find use with the present invention can includebut not be limited to sugars, lectins, antigens, intercalators,chelators, biotin, digoxygenin and combinations thereof. The particularchoice of a dye used to synthesize a novel labeling reagent of thepresent invention may depend upon physical characteristics such asabsorption maxima, emission maxima, quantum yields, chemical stabilityand solvent solubility. A large number of fluorescent andchemiluminescent compounds have been shown to be useful for labelingproteins and nucleic acids. Examples of compounds that may be used asthe dye portion can include but not be limited to xanthene, anthracene,cyanine, porphyrin and coumarin dyes. Examples of xanthene dyes that mayfind use with the present invention can include but not be limited tofluorescein, 6-carboxyfluorescein (6-FAM), 5-carboxyfluorescein (5-Fam),5- or 6-carboxy-4, 7,2′, 7′-tetrachlorofluorescein (TET), 5- or6-carboxy-4′5′2′4′5′7′ hexachlorofluorescein (HEX), 5′ or6′-carboxy-4′,5′-dichloro-2,′7′-dimethoxyfluorescein (JOE),5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein (ZOE) rhodol, rhodamine,tetramethylrhodamine (TAMRA), 4,7-dichlorotetramethyl rhodamine(DTAMRA), rhodamine X (ROX) and Texas Red. Examples of cyanine dyes thatmay find use with the present invention can include but not be limitedto Cy 3, Cy 3.5, Cy 5, Cy 5.5, Cy 7 and Cy 7.5. Other dyes that may finduse with the present invention can include but not be limited to energytransfer dyes, composite dyes and other aromatic compounds that givefluorescent signals. Chemiluminescent compounds that may be used in thepresent invention can include but not be limited to dioxetane andacridinium esters It should also be understood that ligands and dyes arenot mutually exclusive groups. For instance, fluorescein is a well knownexample of a moiety that has been used as a fluorescent label and alsoas an antigen for labeled antibodies.

[0126] The reactive group of the novel labeling reagents of the presentinvention is chosen from chemical moieties that are known to be able toparticipate in carbon-carbon bond formation thereby allowing the novellabeling reagent to attach a label to a suitable target molecule.Examples of such reactive groups can comprise but not be limited toalkenes, alkynes, metallo-organic compounds and halogenated compounds.The metallo-organic and halogenated compounds can comprise aromatic,heterocyclic, alkene, and alkyne groups as well as various combinationsthereof. Although such groups have been described previously forsynthesis of labeled compounds, these reactive groups were only used inthe context of adding amino groups to nucleic acids in order to makenucleotides and polynucleotides look like proteins (U.S. Pat. No.4,711,955 and U.S. Pat. No. 5,047,519, both of which are incorporated byreference). In the present invention, the reactive group of the novellabeling reagent can be attached directly to a ligand or dye, at theterminal end of a linking arm or at an internal site within a linkingarm. A review of various methods for use of metallo-organic andhalogenated compounds is given by Larock (1982, Tetrahedron Report 128;1713-1754), Robins et al. (J. Org Chem 1983, 48; 1854-1862), Hobbs andCocuzza (U.S. Pat. No. 5,047,519), Eglinton and McCrae (1963, Advancesin Organic Synthesis 4; 225-328) and Rieke (2000, Aldrichimica Acta 33;52-60) all of which are incorporated by reference.

[0127] A linking arm that comprises a portion of the novel labelingreagents can be of any desired length and can be comprised of anysuitable atoms that can include but not be limited to carbon, nitrogen,oxygen, sulfur and any combination thereof. Chemical groups that cancomprise the linker arm can include but not be limited to aliphaticbonds, double bonds, triple bonds, peptide bonds, aromatic rings,aliphatic rings, heterocyclic rings, ethers, esters, amides, andthioamides. The linking arm can form a rigid structure or be flexible innature.

[0128] The present invention may be used to label a large variety oftarget molecules. The targets may intrinsically comprise chemicalmoieties that can participate in formation of a carbon-carbon bond withthe reactive group of the novel labeling reagent or the targets may bemodified such that they comprise such a group. Examples of chemicalmoieties on target molecules that can combine with the reactive group ofthe novel labeling reagent can comprise but not be limited to alkenes,alkynes, metallo-organic compounds and halogenated compounds. Themetallo-organic and halogenated compounds can comprise aromatic,heterocyclic, alkene and alkyne groups as well as various combinationsthereof. Target molecules that may find use with the present inventioncan include but not be limited to nucleotides, oligonucleotides,polynucleotides, peptides, oligopeptides, proteins, ligands, syntheticcompounds, synthetic polymers, saccharides, polysaccharides, lipids andhormones. Nucleotides that can be labeled by these compounds can includebut not be limited to monophosphates, diphosphates or triphospates. Theymay be ribonucleotides or deoxynucleotides. Modified nucleotides orNucleotides analogues of any of the foregoing may also be used ifdesired. Examples of modified nucleotides can include but not be limitedto dideoxy nucleotides and nucleotides with 3′ amino or 3′ phosphategroups. Examples of nucleotide analogues can include but not be limitedto peptide nucleic acids, arabinosides, and acyclo versions. Theseanalogues may be used as nucleotides or as components ofoligonucleotides or polynucleotides. Synthesis of a labeledoligonucleotide or polynucleotide can be carried out by the use ofnucleotides that have been labeled by the novel labeling reagent.Alternatively, modified nucleotides that have chemical groups that canbe used for carbon-carbon bond formation with the novel labelingreagents can be used to synthesize oligonucleotides or polynucleotides.In this method, the presence of reactive groups in the oligonucleotideor polynucleotide products allows a subsequent reaction with the novellabeling reagents of the present invention. Additionally, unmodifiedoligonucleotides and polynucleotides can be chemically treated such thatthey comprise groups capable of participating in carbon-carbon bondformation.

[0129] Attachment of the novel labeling reagents of the presentinvention to desirable target molecules can be carried out by any of avariety of means known to those skilled in the art. For instance, theacetoxymercuration reaction is a well known and established procedurefor introducing covalently bound mercury atoms onto the 5-position ofthe pyrimidine ring or the C-7 position of a deazapurine ring (Dale etal., (1975) Biochemistry 14, 2447-2457, Dale et. al. (1973) Proc. Natl.Acad. Sci. USA 70; 2238-2242. The nucleotides are treated with themercuric acetate in sodium acetate buffer to convert them into mercuricsalts. In the presence of K₂PdCl₄, the addition of a labeled reagent ofthe present invention that has been prepared with a terminal double bondwill allow a carbon-carbon double bond to be formed between the aromaticring of the nucleotide and the terminal carbon of the double bond of thelabeling reagent thereby attaching the label to the nucleotide. In thecase of novel labeling reagents of cyanine dyes with double bonds at theteminus of a linker, the mercuric nucleotide reacts with the double bondat the terminus of the linker arm rather that the aromatic ring or theconjugated double bond between the two rings of the cyanine dye moiety.

[0130] In an alternative use of the reaction described above, a novellabeling reagent of the present invention can be prepared where thereactive group is a mercury salt. This compound can now react with anunsaturated bond on the target that is desired to be labeled. This bondmay be an intrinsic part of the target molecule or the target moleculemay be modified to include such a group. Reactions can also be carriedout where both the labeling reagent and the target molecules comprisemercury salts. For instance, Larock (1982 op. cit. Eqns. 146-151) hasdescribed how two groups that each have the structure R1—C═C—HgCl can bejoined together in the presence of appropriate catalysts.

[0131] One advantage of the mercuration and palladium catalyzed additionreactions is that they can be carried out in water with a small amountof organic solvent added to increase solubility if necessary. Thisprocedure can be carried out with the nucleotides in any form, forexample with ribonucleotides, deoxynucleotides, dideoxynucleotides andany analogue, as well as with oligonucleotides, oligonucleotideanalogues, protein nucleic acid complexes and polynucleotides.Alternatively the novel labeling reagent can be prepared with a reactivearm containing a terminal triple bond or any other substance which iscapable of forming the carbon-carbon double bond with the targetmolecule.

[0132] 2. Labeling Proteins by Carbon-Carbon Bond Formation

[0133] An important use for the novel labeling reagent may also be forattaching signal-groups to proteins. In this particular case,modifications of the protein can be made to make them resemble thenucleic acid target described above. For instance, a target protein canbe reacted with mercuric acetate thereby forming mercurate compounds attyrosine, tryptophan or phenylalanine residues in the protein. Theprotein is now available for reacting with a novel labeling reagent thathas an double bond reactive group where displacement of the mercury willtake place while attaching the label. If desired, thiol groups in theprotein can be protected on the protein by treatment with 2,2′-dipyridyldisulfide prior to the mercuration step.

[0134] Amino acids that have primary amines are also sites on a proteinthat may be used with the novel labeling reagent. For instance, proteinsthat lack tyrosine groups can be modified with Bolton-Hunter activeester to introduce tyrosine groups onto primary amines. These can thenbe subsequently used as described above. Alternatively, the protein canbe modified with acrylic acid active ester to introduce terminal doublebonds into residues that contain primary amines. This modification wouldallow proteins to be used with novel labeling reagents of the presentinvention that have mercurate compounds as reactive groups.

[0135] 3. Dye Precursors with Reactive Groups for Carbon-Carbon BondFormation

[0136] Attachment of a group to a marker that is suitable forparticipating in a carbon-carbon bond can be carried out by modificationof the marker. On the other hand, attachment can take place with anintermediate that is used to synthesize a particular marker. Forexample, cyanine dye labeling reagents have the following structure:

[0137] where n=1, 2 or 3; X1 and X2 can be S, O, N, CH₂ or C(CH₃)₂ andR1-R8 comprises reactive group that could be used to join the cyaninedye to a desirable target molecule. Cyanine dyes are prepared by linkingtogether two indolenine precursor units with an intervening unsaturatedchain. The particular number of units making up the chain determines theparticular absorption and emission spectra of the cyanine dye.

[0138] According to the method of the present invention, cyanine dyescan be prepared by attaching a linker arm containing a reactive groupcapable of generating a carbon-carbon bond to the indolenine ring thatis a precursor to a cyanine dye. This modified indolenine is a novelcompound that can then be used as a reagent in a reaction where it iscoupled to a second indolenine ring through an intervening unsaturatedalkyl chain to synthesize a cyanine dye with the structure describedabove. The second indolenine can be the same as the first or it may bean unmodified version that lacks the linker arm and reactive group. Thesame novel indolenine compound can be used to make a variety ofdifferent cyanine dyes depending upon the nature of the secondindolenine ring and the particular unsaturated chain joining the twoindolenine rings. As a result of this procedure, when the cyanine dyeproduct is formed by joining the precursor rings, it already comprises alinker arm with a reactive group and is ready to be attached to asuitable target molecule.

[0139] 4. Novel Rhodamine Dyes Without the Phenyl Group

[0140] In another aspect of the present invention, new dyes and meansfor their synthesis are disclosed. In previous art, derivatives ofrhodamine typically have an aromatic group between the dye and thereactive group that is used to attach the rhodarine to a desirablemolecule. In the present invention, it is disclosed that stablenucleotides can be synthesized that comprise rhodamine analogues wherethe linker arm joining the dye to a base on the nucleotides lacks thearomatic group that is normally present in rhodamine. It is a surprisingconsequence that incorporation of such nucleotides becomes moreacceptable to a polymerase such that the modified nucleotide can be usedwithout mixing it with unmodified nucleotides. As such, the presentinvention discloses the following novel rhodamine analogues:

[0141] where R is a reactive group.

[0142] 5. Rigid Linker Arms

[0143] In another aspect of the present invention, methods andcompositions are disclosed that enable modified nucleotides to be usedmore efficiently in enzymatic and chemical means of synthesis and/orallow them to function more efficiently when they are part of apolynucleotide. In one embodiment of the present invention, an increasedand directed separation is achieved between a target molecule and thegroup that has been added to provide a marker or label. In the presentinvention,:the following novel compositions are disclosed that have theformulas:

T—L—M and R—L—M

[0144] In the diagram above, T is a target for attachment of a marker orlabel; R is a reactive group that may be used for attachment to a targetand M is a marker or label.

[0145] In one aspect of the present invention, L is a chemical groupthat covalently connects the M moiety to the T moiety or R moiety andcomprises one or more of the following groups:

[0146] The alkene groups can be in either cis or trans configurationwith regard to each other and they may comprise only hydrogen atomsbonded to the carbon atoms or they may be substituted. In a preferredmode, directionality is derived from having one of the groups aboveimmediately linked to the target, separated by no more than a singleintervening atom or linked to the target through a rigid polar unit.

[0147] In another aspect of the present invention, L is a chemical groupthat covalently connects the M moiety to the T moiety and comprises atleast two consecutive rigid polar units.

[0148] Examples of targets that may find use in the present inventioncan include but not be limited to nucleotides, oligonucleotides,polynucleotides peptides, polypeptides, cytokines, ligands, haptens,antigens and solid supports. Examples of solid support that may find usewith the present invention can include but not be limited to beads,tubes, plastic slides, glass slides, microchip arrays, wells anddepressions.

[0149] Examples of reactive groups that may find use with the presentinvention can include but not be limited to isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono or di-halogen substitutedpyridine, mono or di-halogen substituted diazine, aziridine, sulfonylhalide, acid halide, hydroxy-succinimide ester, hydroxy-sulfosuccinimideester, imido esters, glyoxal groups, aldehydes amines, sulfhydrylgroups, hydroxyl groups. Also included are groups that can participatein carbon carbon bond formation as disclosed previously.

[0150] In the present invention, a rigid unit is defined as a group ofatoms where the spatial relations between the atoms are relativelystatic.

[0151] In the present invention, when two moieties are described asconsecutive, the moieties are adjacent or directly next to each other.Additionally, two consecutive moieties can be separated by no more thanone atom, i.e. a single atom.

[0152] In the present invention, a rigid unit is non-polar when itessentially comprises the same type of atoms. Examples of non-polarrigid units would be alkenes, alkynes, unsaturated rings, partiallysaturated rings and completely saturated rings that comprise only carbonand hydrogen.

[0153] In the present invention, a rigid unit is polar when it comprisesat least two or more different atoms thereby distributing the chargeunequally through the unit. Examples of an arrangement that couldcontribute to polarity could include but not be limited to a carbon atomthat is bonded to N, S, O, P, or a halogen. The heteroatoms that arebonded to the carbon may be used alone or they may be part of polar orcharged functional groups. Examples of the latter can include but not belimited to —OH, —SH, —SO₃, —PO₄, —COOH and —NH₂ groups. The rigid unitscan comprise backbones that are linear, branched or in ring form. Thering forms that also comprise polar or charged functional groupsattached to the rings may be unsaturated rings, partially saturatedrings and completely saturated rings. Multimers of two or more suchpolar rigid units will provide rigid extended arms that create a definedspatial relationship between a target molecule and a marker or signalgenerating moiety.

[0154] In the present invention, unsubstituted heterocyclic aromaticcompounds would be considered to be non-polar rigid units due to theelectron sharing in the ring. On the other hand, substitutedheterocyclic aromatic compounds that comprise polar or chargedfunctional groups attached to the rings would be considered to be polarrigid units.

[0155] Examples of linear polar rigid units that would be useful in thepresent invention can include but not be limited to moieties comprisingpeptide bonds. Examples of cyclic polar units that have inherentrigidity can include but not be limited to sugars. Examples of groupsthat would have utility in the present invention that have rigidityderived from the interactions between subunits can include but not belimited to charged components where charge repulsions can maximizedistances between subunits. When negatively charged components are used,there can also be repulsion away from the negatively chargedpolynucleotide itself. The linker may also be designed with bulky sidegroups that interfere with rotational changes thereby maintaining adiscrete spatial structure with regard to the relationship of a base anda signal or reactive group.

[0156] The distance of the reactive group or signal moiety from thetarget molecule would be determined by the number and nature of therigid units making up the spacer. Thus, a series of three rigid unitsthat comprise an alkene bond followed by two peptide bonds would extendthe signal group directly away from the nucleotide as shown in FIG. 1A.This particular example would comprise a non-polar rigid unit as well astwo polar rigid units. A series of multiple peptide bonds could stillprovide rigidity while extending the dye or marker further away from thetarget molecule as shown in FIG. 1B. In this particular illustration auracil nucleoside is used as a target and glycine subunits are used toprovide a series of peptide bonds. Different amino acids may also havebeen used if so desired, where the various constituents of the R groupsof the amino acids may be chosen to endow other properties such assolubility or charge upon the rigid arm.

[0157] A linker that is comprised of rigid units will depend upon theparticular relationship between the rigid units for whether the overallstructure is rigid or not. For instance, multiple peptide bonds havebeen used in prior art. However, the beneficial qualities of having suchbonds were lost by the inclusion of aliphatic carbon groups in betweenthe peptide. In essence, these were rigid units joined by flexiblelinkers. As seen in FIG. 1, the present invention allows for at most asingle atom between the rigid units, thereby limiting the extent offlexibility between rigid units. Similarly other groups of a non-carbonnature could be used between groups that would retain an overallrigidity while contributing a potentially desirable directionality. Anillustrative example of such a group would be a —S— bond between tworigid units.

[0158] As described above, sugar groups may also be used in carrying outthe present invention. There are a wide variety of sugars that can beused as individual rigid units and a large number of ways that thesesugars can be linked together either enzymatically or chemically hasbeen extensively described in the literature.

[0159] Although the present invention makes use of two or more polarrigid units to create a rigid linker arm, it is understood that flexiblegroups and non-polar rigid units may also be included in the rigidlinker arms. For instance, FIG. 1 makes use of an alkene bond betweenthe peptide bonds and the uracil moiety. In addition, additionalflexible units, rigid units or combinations thereof may be includedbetween the last peptide bond and the dye molecule in FIG. 1 whileretaining the effectiveness of the linker.

[0160] In the present invention, the presence of such an extendedlinkage away from a nucleotide should decrease deleterious effects uponincorporation since the problematic group should be spatially displacedfrom the active site where enzymatic incorporation is taking place. Inaddition, after a modified nucleotide is incorporated eitherenzymatically or synthetically, functionality may also be increased bythe use of the present invention. For instance, extension of a hapten ora chemically reactive group further from an oligonucleotide orpolynucleotide should provide increased accessibility thereby improvingbinding or coupling efficiencies. In addition, signal generation groupscould also be displaced away from the oligonucleotide or polynucleotideby the use of the present invention if interference effects are causedby proximity.

[0161] The particular point of attachment of the linkers described inthe present invention may take advantage of previously described art forflexible linkers. As such, the nucleotides may be normal nucleotides orthey may be modified nucleotides or nucleotide analogues with varioussubstituents either added or replacing components in the base, sugar orphosphate moieties as disclosed in U.S. Pat. No. 4,711, 955; U.S. Pat.No. 5,241,060; U.S. Pat. No. 4,952,685 and U.S. Pat. No. 5,013,831; allof which are hereby incorporated by reference). In addition, thesemodifications may be non-disruptive, semi-disruptive or disruptive. Thepoint of attachment may be the base, sugar or phosphate as described inthe previously recited disclosures, but attachment to the base isparticularly useful in the present invention.

[0162] A further benefit of the present invention is that some of thelinkers that have been described may offer beneficial results due totheir chemistry as well as structure. For instance, the last peptide ina linker composed of peptide subunits offers an amine group that may beused to attach useful groups such as signal moieties. In prior art,amine groups were located at the ends of aliphatic chains, with pKvalues of about 11. However, since coupling reactions are usuallycarried out at around pH 8 values, very little of the amine group is ina reactive form at any given time, thereby limiting the efficiency andkinetics of the reaction. In contrast, the amine group at the end ofpeptide chain has a pK of about 9, a value that is more compatible withthe intended coupling reaction. Thereby, the present invention allowsmore effective coupling of a nucleotide or polynucleotide to anappropriate group.

[0163] Also, although this particular aspect of the present inventionhas been described in terms of a rigid linker intervening between anucleotide and a dye, other applications may also enjoy the benefits ofthe present invention. For instance, labeling of proteins can beimproved by using the rigid arm of the present invention between theprotein and a signal moiety. Examples of proteins that might enjoy thesebenefits can include but not be limited to antibodies, enzymes,cytokines and libraries of oligopeptides or polypeptides. As describedpreviously for nucleic acids, the use of the present invention mayimprove the properties of the labeled compound as well as the efficiencyof the labeling itself. Additionally, there are many procedures thatinvolve fixation of a ligand, hapten, protein or a nucleic acid to asolid support. Examples of such supports can include but not be limitedto beads, tubes, microtitre plates, glass slides, plastic slides,microchip arrays, wells and depressions. The present invention can beused to generate a directed separation of a ligand, hapten, protein ornucleic acid away from the surface of the support. Examples of proteinsthat might enjoy these benefits can include but not be limited toantibodies, enzymes, cytokines and libraries of oligopeptides orpolypeptides.

[0164] 6. Non-Metallic Porphyrins with Reactive Groups on Non-PyrrolePositions

[0165] In another aspect of the present invention, a novel labelingreagent is disclosed that comprises a non-metallic porphyrin with areactive group at a non-pyrrole position. The spectral quality ofnon-metallic alkylated porphyrins as fluorescent dyes has been describedin Hendrix in U.S. Pat. No. 4,107,454 (incorporated by reference) whereStokes shifts over 150 nm were disclosed. However, when describingreactive groups for the porphyrins, the only teachings that weredisclosed made use of chemical groups on the pyrrole positions.Therefore, it is a subject of the present invention that non-metallicporphyrins can be derived which independently comprise hydrogen,aliphatic, unsaturated aliphatic, cyclic, heterocyclic, aromatic,heteroaromatic, charged or polar groups on any or all of the eightpyrrole positions and use one of the non-pyrrole positions as a site forattaching a reactive group. This composition has the followingstructure:

[0166] where R₀ comprises a reactive group attached directly orindirectly to a non-pyrrole position of the porphyrin (i.e., the α, β,γ, or δ positions) and R₁ through R₈ are as defined just above.

[0167] Any of the reactive groups that have been described previouslymay find use in the present invention as R₀. R₁ through R₈ may comprisethe same groups or they may be different. If desired, the alkyl groupsmay also further comprise polar or charged groups that may aid inincreasing the aquaeous solubility of the porphyrin. Also if desired,there may be a linker used to attach a reactive group to the porphyrin.,The particular rigid arm used in this aspect of the present inventioncan be any linker arm that has been previously disclosed or describedbut it is especially preferred that the rigid linker of the presentinvention be used. A nitro group can be added to a non-pyrrole positionas described by Fuhrhop and Smith “Laboratory Methods” Chapter 19 inPorphyrins and Metalloporphyrins, Kevin M. Smith, editor, ElsevierScientific Publishing Company, Amsterdam, 1975, hereby incorporated byreference. Reduction of this group to an amine is well known to thoseskilled in the art and further reaction can be carried out to add alinker or a reactive group by standard techniques.

[0168] Any of the previously described targets may be labeled by thenon-metallic porphyrins of the present invention. For example, thenon-metallic phorphyrins of the present invention may find use byincorporation of a porphryin labeled nucleotide or synthetically by aporphyrin labeled phosphoramidite. Alternatively, oligonucleotide orpolynucleotides can be synthesized that have derivatived nucleotidesthat are suitable for reaction with a chemically compatible derivativeof a non-metallic porphyrin in a post-synthetic step. Labeledoligonucleotides and polynucleotides that comprise the non-metallicporphyrin of the present invention should enjoy a large Stokes shiftwith high efficiency emission. This composition and method of detectionwill enjoy a high level of sensitivity as well as enabling high level ofdiscrimination from other compounds that may be excited at the samewavelength. For instance, if a library of transcripts is labeled withfluorescein and a second library is labeled with octaethylporphine,illumination can be carried out by a single wavelength of 490 nm. Yet,discrimination between the particular fluorophores is easilydistinguishable since the emission peak is 530 nm for fluorescein andthe emission peak for octaethylporphine is 620 nm. At the same time, thequantum yield for the octaethylporphine is comparable to that offluorescein. It is also understood that the non-metallic phorphyrins maybe used in conjunction with any of the other novel methods that aredisclosed herein.

[0169] 7. Modification of Dyes by Groups that Participate in theConjugation and/or Electron Delocalized System

[0170] In another embodiment of the present invention, methods aredisclosed for the synthesis of novel compositions that comprise two ormore unsaturated compounds added to a fluorescent dye without arequirement for the presence of kenone groups in an intermediate. In thepresent invention, these unsaturated compounds can be unsaturatedaliphatic groups, unsaturated cyclic compounds, unsaturated heterocycliccompounds, aromatic groups or any combinations thereof. Attachment ofsuch groups allows them to participate in the conjugation and/or theelectron delocalized system (Maulding and Roberts, op. cit. incorporatedby reference) of the dye and confer changes upon the spectralcharacteristics of the dye. These changes can include changing the widthof the excitation and emission peaks, shifting the positions of theexcitation and emission peaks and increasing the quantum yield.

[0171] Since addition of unsaturated groups to the dyes may decreasesolubility or result in non-specific hydrophobic interactions, it is anobjective of the present invention that this effect can be compensatedby a further addition of charged or polar groups. These may be attachedto the dye or to the unsaturated compounds. Also, since these novel dyesfind use as labeling reagents, they may also comprise reactive groupssuitable for attaching the label to desirable target molecules. Thereactive groups can be directly or indirectly linked to the dye, to theunsaturated compounds or to the charged or polar modification groups.

[0172] The novel composition of this aspect of the present invention hasthe following structure:

R-Dye

[0173] where Dye is a fluorescent dye; R is covalently linked to the Dyeand R comprises two or more unsaturated compounds which can beunsaturated aliphatic groups, unsaturated cyclic compounds, unsaturatedheterocyclic compounds, aromatic groups or combinations thereof.Furthermore, one or more members of R participates in the conjugationand/or electron delocalized system of the Dye. The unsaturated compoundscan be substituted or unsubstituted. The unsaturated aliphatic group cancomprise an alkene or an alkyne. The aromatic group can comprise aphenyl group, an aryl group, or an aromatic heterocycle. When the groupsare substituted, the substituents can include but not be limited toalkyl groups, aryl groups, alkoxy groups, phenoxy groups, hydroxylgroups, amines, amino groups, amido groups, carboxyl groups, sulfonates,sulfhydryl groups, nitro groups, phosphates or any group that canimprove the properties of the dyes. In the case of an aromatic group, itmay also be substituted by being part of a fused ring structure.Examples of such fused rings can include but not be limited tonaphthalene, anthracene, and phenanthrene.

[0174] Groups that can be used as all or part of R can also be describedas follows:

[0175] In the diagram above, Ar is an unsaturated cyclic compound, anunsaturated heterocyclic compound or an aromatic group. As describedpreviously, the groups above may be substituted or unsubstituted.

[0176] Fluorescent dyes that may find use with the present invention caninclude but not be limited to anthracene, xanthene, cyanine, porphyrin,coumarin and composite dyes. In the case where anthracene is used as theDye with an alkyne joined to the center ring and a phenyl group attachedto the alkyne, the phenyl group will be substituted.

[0177] In another aspect of the present invention, a novel compositionhas the following structure:

[R-Dye]—R₁

[0178] where R and Dye are as described previously and where R₁ iscovalently joined to R, Dye or to both R and Dye. R₁ further comprisesone or more charged or polar groups to provide additional solubility.This may useful when the dye or the dye with the R modification haslimited aquaeous solubility or problems with non-specific hydrophobicinteractions.

[0179] In another aspect of the present invention, a novel compositionhas the structure

[0180] where R, Dye and R₁ are as described previously and where R₂ iscovalently attached to R, Dye, R₁ or any combination thereof and whereR₂ further comprises a reactive group that can be used to attach the dyeto a suitable target molecule. R₂ can comprise any of the reactivegroups previouly described including sulfhydryl, hydroxyl and aminegroups, groups capable of reacting with sulfhydryl, hydroxyl and aminegroups, and groups capable of forming a carbon-carbon bond. R₂ canfurther comprise a linker arm that that separates the reactive groupfrom the dye. The linker arm can be of any desirable length and cancomprise a backbone of carbon as well as non-carbon atoms. Non-carbonatioms that may find use can include but not be limited to sulfur,oxygen and nitrogen. The linker arm can comprise saturated, unsaturatedor aromatic groups and may also comprise the rigid arms describedpreviously.

[0181] In another aspect of the present invention, a novel labeledtarget comprises the structure:

[R-Dye]—target

[0182] where R and Dye are as described previously. Targets that mayfind use with the present invention can include but not be limited to aprotein, a peptide, a nucleic acid, a nucleotide or nucleotide analog, areceptor, a natural or synthetic drug, a synthetic oligomer, a syntheticpolymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, anantigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- orpolysaccharide. The labeled target can also further comprise R₁covalently joined to R, Dye or to both R and Dye. R₁ further comprisesone or more charged or polar groups to provide additional solubility.This may useful when the labeled target or an intermediate used formaking the labeled target has limited aquaeous solubility or problemswith non-specific hydrophobic interactions. The labeled target can alsofurther comprise a linker arm as described above separating the dye fromthe target.

[0183] 8. Intercalators

[0184] In another aspect of the present invention, a novel method isdisclosed that provides enhanced discrimination between an intercalatingdye that is bound to a target compared to dye that remains unbound. Asdescribed previously, ethidium bromide has been a popular reagent fordetection and visualization of DNA in a number of formats. Toinvestigate the effect of a second-phenanthridinium ring system onaffinity to nucleic acids, a homodimeric form of ethidium bromide wassynthesized and tested (Kuhlmann et al., (1978) Nucleic Acids Research,5; 2629-2633). This compound,N,N-Bis[3-(3,8-diamino-5-methylphenanthridinium-6-yl)benzoyl]1,5-diaminopentane dichloride (meta-EthD) exhibited a muchhigher affinity to nucleic acids than the monomeric form. However, whenfluorescence was measured at the standard wavelength of 493 nm, theincrease in fluorescent emission after binding to nucleic acids wasessentially the same as seen earlier for the ethidium bromide monomer.

[0185] It was a surprising and unexpected result that when meta-EthD wasused in a different manner than the standard format, a greatly enhanceddiscrimination between bound and unbound was observed. The presentinvention discloses that when two ethidium bromide molecules are joinedtogether through their phenyl groups, excitation at a wavelength below400 nm can result in an increase of over 150 fold in fluorescentemission upon the binding of DNA to the homodimer as opposed to the 6fold increase seen when the samples are excited at 493 nm.

[0186] Two other homodimeric ethidium bromide compounds (EthD-1 andEthD-2) are commercially available from Molecular Probes, Inc. (Eugene,OR). However, in contrast to the results with meta-EthD,-thediscrimination between bound and unbound dye was not substantiallychanged by exciting at wavelengths below 400 nm. It should be pointedout that although meta-EthD, EthD-1 and EthD-2 are all ethidium bromidedimers, they are chemically dissimilar. As shown in (FIG. 2), meta-EthDis comprised of two phenanthridinium rings linked together through themeta position of the phenyl rings through amide bonds. In contrast, thephenanthridinium rings of EthD-1 and EthD-2 dimers are joined togetherthrough the nitrogen of the center rings rather than through the phenylrings. The intervening chain is comprised of an alkyl chain with twoamine attachment groups which are secondary in EthD-1 and methylated togive the quaternary salts for EthD-2. The inability of the EthD-1 andEthD-2 compounds to exhibit the same results seen with meta-EthDdemonstrates that the method of the present invention was not apredictable property of ethidium dimers per se.

[0187] The method of the present invention may find use in many methodsthat had been previously described for ethidium bromide, ethidiumbromide homodimers and other intercalators. Of especial use, is theapplication of the present method towards real time analysis of nucleicacid amplification and probes labeled with meta-EthD. The large increasein fluorescence after illumination at wavelengths under 400 nm willallow a better signal to noise ratio than previous methods. Thereby, thepresent invention should enjoy a higher sensitivity of detection of thesynthesis of nucleic acids during such amplification procedures.

[0188] In previous art, ethidum bromide has also been modified throughthe center ring by attaching other intercalators (U.S. Pat. No.5,646,264) and fragments of intercalators (U.S. Pat. No. 5,582,984 andU.S. Pat. No. 5,599,932) for improved performance of binding todouble-stranded DNA. Modification groups similar to those disclosed inU.S. Pat. No. 5,582,984 have also been added to the central ring ofethidium bromide to improve performance with RNA (U.S. Pat. No.5,730,849). In light of the results with the meta-EthD, it is disclosedthat the modifications in U.S. Pat. Nos. 5,646,264; 5,582,984;5,599,932; and 5,730,849; all of which are incorporated by reference,may also be used to synthesize novel compounds by replacing the centerring with the phenyl ring as an attachment site. These may also be usedin many of the applications previously described for ethidum bromide,ethidium bromide dimers, ethidum bromide heterodimers, modified ethidiumbromide compositions and other intercalators.

[0189] 9. Novel Chemiluminescent Reagents

[0190] In another embodiment of the present invention, novel1,2-dioxetanes compounds are disclosed that when used as substrates forselected enzymes result in an intramolecular reaction between two groupsattached to different sites of an aromatic ring thereby leading tochemiluminescent signal generation. In another aspect of the presentinvention, the novel 1,2-dioxetanes compounds are disclosed that aresubstrates for modification enzymes rather than degradative enzymeswhere the modification event can lead to chemiluminescent signalgeneration.

[0191] a. Enzyme Dependent Interactions Between Two Groups Attached toDifferent Sites on a Cyclic Ring

[0192] In another aspect of the present invention, novel 1,2-dioxetanereagents are disclosed that comprise two groups attached to differentsites of a cyclic ring where after catalysis by an appropriate enzyme,the reagent undergoes an intramolecular reaction thereby leading tochemiluminescent signal generation. The reagents of this aspect of thepresent invention have the structure:

[0193] where Q comprises a cycloalkyl or polycycloalkyl group located onone side of the dioxetane and R1 and R2 are located on different sitesof a cyclic ring that is bonded to the other side of the 1,2-dioxetane.Z can comprise hydrogen, alkyl, aryl, alkaryl, heteroalkyl, heteroaryl,cycloalkyl, or cycloheteroalkyl groups. In a preferred embodiment, Qcomprises an adamantyl group. In another preferred embodiment, the twosites where R1 and R2 are attached are adjacent to each other on anaromatic ring. R1 comprises a chemical group that is a substrate for anenzymatic activity. In the presence of the appropriate enzyme, R1 iscatalytically converted into R1* which comprises a chemically reactivegroup G1. R2 is attached to the ring through an oxygen atom andcomprises a chemical group G2 that is capable of interacting with the G1group that is produced by the conversion of R1 into R1*. Due to therigidity imparted by the ring, G1 is in close proximity to G2 therebyendowing the interaction to take place with favorable kinetics. Thisinteraction leads to formation of an unstable dioxetane therebyproducing chemiluminescence.

[0194] R2 can comprise an aliphatic group, substituted aliphatic group,an aromatic group or any combination of the foregoing. In the caseswhere R2 comprises a substituted aliphatic group, the substituents canbe halogens, nitrate, sulfur or nitrite. The aliphatic group can besubstituted at one position or in several positions. The substituents ateach position can be the same or different.

[0195] As described above, after the enzymatic conversion of R1 into R1*a chemically reactive group G1 is formed. Chemically reactive atoms thatmay find use as part of G1 may include but not be limited to nitrogen,sulfur or oxygen. Enzymes that may find use with present invention caninclude but not be limited to amidases, esterases,acetylcholinesterases, acid and alkaline phosphatases, decarboxylases,lipases, glucosidase, xylosidase, fucosidase, trypsin and chymotrypsin.Enzymatic substrates that may find use as constituents of R1 can includebut not be limited to amides, esters, phosphates, carboxylic acid, fattyacids, glucose, xylose, fucose or amino acids.

[0196] Although it is not essential, R1 can also be designed such thatafter the enzymatic conversion of R1 into R1* the interaction between G1and G2 creates a 5 or 6 membered ring which is known to be an especiallystable conformation. An example of formation of such an intermediate isshown below using oxygen as the connection of R2 to an aromatic ring:

[0197] As shown above, the intermediate structure can undergo aninternal substitution reaction that transfers the G2 group to the G1group of the R1* moiety thereby releasing the oxygen and creating anunstable phenoxy ion leading to an unstable form of dioxetane andproduction of a chemiluminescent signal. The juxtaposition of the G1 andG2 groups caused by locating each group on a segment of a rigidstructure should allow efficient interaction and subsequentsubstitutions and rearrangements to form the light producingintermediate after production of G1 by enzymatic activity.

[0198] b. Chemiluminescence Generation Derived from Modification Enzymes

[0199] In another aspect of the present invention novel 1,2-dioxetanederivatives are disclosed in which the triggering event that leads tothe decomposition and production of chemiluminescent signal is an enzymemodification of a specific group of the structure. This is in directcontrast to previous examples in which the triggering event is thecleavage of a substituent. In a preferred mode, the modification of thesubstitutent is dependent upon an enzymatic reaction. An example of sucha composition is given below:

[0200] In the diagram above, Q and Z are defined as described previouslyand R can comprise a chain of atoms consisting of C, N, O, S or anyother atoms required. R can also comprise saturated or unsaturatedgroups. Furthermore, R′ can include but is not limited to alcohols orcarboxylic groups. The modification reaction that can lead to lightproduction reaction can include but not be limited to oxidation andreductions,. Enzymes that can be used in this aspect of the presentinvention can include but not be limited to oxidases and reductases.

[0201] A representative example of this process is given below where adioxetane derivative comprising a terminal alcohol is enzymaticallyconverted to an aldehyde by the action of alcohol dehydrogenase.

[0202] This resulting product can then undergo t elimination that thenresults in the unstable phenoxide ion that triggers the decomposition ofthe 1,2-dioxetane resulting in the chemiluminescent signal.

[0203] 10. Real-Time Signal Generation

[0204] The present invention discloses a method of signal generationthat can be used for labeling either discrete nucleic acids or a libraryof multiple sequences. The present invention provides methods andcompositions for specifically labeling analytes of interest in thepresence of other nucleic acid sequences. The present invention may alsobe used for the detection of the presence and/or amount of nucleic acidsof interest during the course of using such nucleic acids as templatesfor further nucleic acid synthesis. This can be carried out either bypost-synthesis analysis or real-time analysis during the course of suchsynthesis. In the present invention, nucleic acids are synthesized thatcomprise at least one first element of an energy transfer pair and atleast one second element of an energy transfer pair. When a first energytransfer element is capable of acting as an energy donor, the secondenergy transfer element is capable of acting as an energy transferacceptor. Conversely, the first element can be an energy transferacceptor and the second element can be an energy donor. This secondelement comes into association with the first element by virtue ofeither being incorporated into the same nucleic acid strand thatcomprises a first element or by binding to a nucleic acid strand. In theabsence of nucleic acid synthesis or a binding event, there is little orno energy transfer from the donor to the acceptor. However, by theappropriate designs, the present invention allows energy transfer from adonor to an acceptor during or after nucleic acid synthesis.

[0205] Various embodiments of the present invention use labeled primers,probes, nucleotides, nucleic acid binding agents and solid supports assources of energy transfer elements. In the present invention, a probeand a primer share the common characteristic of binding to complementarysequences with the proviso that a primer has the additional property ofbeing able to be extended. Nucleic acid constructs may also be used inthe present invention as primers, probes or templates. In the presentinvention a nucleic acid construct comprises a nucleic acid withsequences that are either identical or complementary to all or a portionof a nucleic acid of interest and may further comprise at least onenon-natural or artificial element.

[0206] Examples of non-natural or artificial elements that couldcomprise a nucleic acid construct can include but not be limited topromoter sequences, capture sequences, identity tag sequences, consensussequences, protein binding sequences, artificial primer bindingsequences, modified nucleotides, nucleotide analogues, abasic sites,labels, ligands, peptides and proteins. Furthermore nucleic acidconstructs may comprise analytes. These analytes can be individualspecific sequences or a library of sequences. They may be the originalanalyte itself or a copy thereof. They can be derived from chromosomes,episomes or fragments thereof. Examples of episomes can include but notbe limited to plasmids, mitochondrial DNA, chloroplast DNA and viruses.

[0207] a. Energy Transfer Between Labeled Primers

[0208] In one embodiment of the present invention, the first and secondenergy transfer elements are components of at least two primers ornucleic acid constructs that can be extended in the presence ofappropriate nucleic acids. At least one of these primers or nucleic acidconstructs will comprise sequences that are complementary to sequencesthat are present in a portion of a nucleic acid of interest. At leastone other primer or nucleic acid construct will comprise sequences thatare identical to sequences that are present in another portion of thenucleic acid of interest. In this way, a nucleic acid of interest can beused as a template for binding and extension of the primer or nucleicacid constructs. Separation or displacement of the extended primer fromthe target allows the target strand to be used for another primerbinding/extension event. In addition the extended primer can itself beused for a primer binding/extension event. Thus one would create aproduct that comprises two extended primers hybridized to each other. Inthis aspect of the present invention, the primers used for the precedingsequential primer binding/extension events comprise either a firstenergy transfer element or a second energy transfer element. If theprimers in each strand are in sufficient proximity to each other, theywould be capable of allowing an energy transfer event from a donor to anacceptor. This process can be used to create a double-stranded labelednucleic acid. Of especial utility for diagnostic purposes, the extent ofthe signal generated by this process can be used to identify thepresence and quantity of the particular nucleic acids used as templates.

[0209] The amount of signal can also be increased by introduction ofamplification processes. For instance, the use of a primer for eachstrand of a desirable nucleic acid target is the basis of many targetamplification procedures where strand extension of each primer generatestemplates for further synthetic events. These methods can depend upondiscrete steps such as those employed in PCR (U.S. Pat. No. 4,683,202)or they can be continuous isothermal methods such as SDA (U.S. Pat. Nos.5,270,184 and 5,455,166) and Loop Mediated Amplification (U.S. patentapplication Ser. No. 09/104,067; and European Patent Application No. EP0 971 039 A) (all the foregoing incorporated by reference). Thus,although the present invention can be used for post-synthesis assessmentof the amount of synthesis of appropriate nucleic acids, it can also beused during the multiple synthetic steps that take place during thecourse of amplification, i.e real time analysis. Amplification can becarried out under the same conditions used in the absence of labeledprimers or an additional step can be included that can increase theefficiency or selectivity of signal generation. For instance, for realtime analysis of an isothermal reaction, monitoring can take placecontinuously or at chosen intervals. In the latter method, an extra stepcan be carried out where either a sample is removed for analysis or athermal step is introduced that promotes signal generation or reading ata particular state but does not substantially interfere with thecontinuation of the reaction.

[0210] In previous art, the design of primer locations for doublestranded synthetic nucleic acid products for diagnostic purposes hasbeen to have the primers for each strand located sufficiently apart thatadditional sequences are incorporated in between them that can be usedfor hybridization with probes or characterization by restrictionenzymes. Thus, the sequences of double stranded synthetic nucleic acidproducts would be derived from two sources. First, there would beintrinsic sequences derived from the primers and their complements.These will be present independent of what particular target segment wasused as a template. Secondly, there would be the sequences between theprimer segments. These would be totally dependent upon the nature of theparticular nucleic acid segment used as a template for nucleic acidsynthesis. Depending upon the nature of the design of the primers, theconditions of the reaction and the particular nucleic acid sequences inthe sample used in the amplification reaction, only a particulardesirable sequence may be synthesized or other non-desirable sequencesmay also be synthesized. For diagnostic purposes, the segments betweenthe primers have then been used as a target for a labeled probe togenerate a signal that would be dependent upon the presence and amountof only the desirable sequences.

[0211] In this particular embodiment of the present invention, therequirement for extended sequences between the primer segments isabrogated since probes are not used for the detection of theamplification product. In fact, the present invention discloses that aproximity between the primers at each end of an amplicon is a desirablearrangement that can be used for a novel means of signal generation. Byincluding a first element into a primer for one target strand and asecond element into a primer for the complementary target strand,proximity of these two primers in a double stranded amplicon allowsenergy transfer to take place from the element that acts as a donor tothe element that acts as an acceptor even though each element is on adifferent strand.

[0212] As described previously, various amplification systems that arebased upon a series of primer extension reactions that result in doublestranded amplicons with incorporated primers will be able to enjoy thisparticular embodiment of the present invention. For instance, FIG. 3shows potential amplification products for a) PCR and b) SDA. Details ofthe processes that can be used for these amplification methods can beseen in numerous publications including the original patent for each ofthese methods (Mullis et al. in U.S. Pat. No. 4,683,202 and Walker etal., in U.S. Pat. Nos. 5,270,184 and 5,455,166; incorporated herein byreference). Even though these methods employ different principles, thepresence of a labeled primer or nucleic acid construct in each strand ofan a double-stranded nucleic acid allows the use of the presentinvention. In addition, other methods that have been previouslydisclosed may find use with the present invention including multiprimeramplification (U.S. patent application Ser. Nos. 08/182,621; filed Jan.13, 1994; U.S. patent application Ser. No. 09/302,816, filed Mar. 31,1998; and U.S. patent application Ser. No. 09/302,818, filed Feb. 3,1998; and U.S. patent application Ser. No. 09/302,817, filed Apr. 16,1999) and amplification with inverted oligonucleotides (U.S. patentapplication Ser. No. 5,462,854) all of which are hereby incorporated byreference.

[0213] As described previously, this particular embodiment of thepresent invention depends upon a proximity between the primers ornucleic acid constructs on each strand. In terms of an extended strandmade from a first primer or nucleic acid construct, this can also bedescribed as the proximity between the segment derived from theincorporated first primer or nucleic acid construct and the segment thatcan be used as a binding site for the second primer or nucleic acidconstruct to synthesize the complementary strand. For instance,proximity can be achieved by having these two segments being immediatelyadjacent to each other on an extended strand. In such a case, thenucleic acid sequences of the extended strands would be entirely derivedfrom the sequences of the primers or nucleic acid construct and theircomplements. To depict this more clearly, an arbitrary sequence is shownin FIG. 4 with potential primer arrangements that could be used in thepresent invention. In FIG. 4(A) the sequences chosen for primers areimmediately adjacent to each other on each strand. Alternatively, therecan be a gap between the primer segment and the primer binding segmenton one strand as long as there is sufficient proximity for energytransfer between the donors and acceptors in the amplification product.An example of a longer spacing using the same target sequence is shownin FIG. 4(B).

[0214] It should also be noted that in addition to allowing a novelsystem of signal generation, the reduction of the amplicon size suchthat it comprises little more than primer binding regions should conferadvantages over the more traditional longer amplicons. These shouldinclude shorter extension times, sharper melting points, and overallhigher efficiency in each round of amplification since the amount ofsynthesis is of a minimal nature. Also, the choice of appropriate energytransfer elements and detection systems can allow multiplexamplification to monitor more than one target sequence.

[0215] In the presence of the appropriate target sequences, signalgeneration should increase as more labeled primers become incorporatedinto double-stranded nucleic acids. This signal generation should bespecific and proportional to the presence of appropriate targetmolecules in the sample. Thus, in the absence of nucleic acid synthesis,there should be little or no energy transfer between donor and acceptormolecules since each element is located on a separate primer or nucleicacid construct. Secondly, signal generation can be carried out underreaction conditions that allow little or no nucleic acid synthesis inthe absence of appropriate target templates. One way that this can becarried out is by appropriate design of the primers themselves such thatprimer-dimer formation is minimized, for instance by selecting primersequences that have no overlap between their 3′ ends. On the other hand,if non-target nucleic acids are present that have sequences presentwhich have some similarity to the primer binding sequences, nucleic acidsynthesis may take place, but the nucleic acid product is unlikely tohave the primers incorporated into the appropriate lengths for energytransfer to take place. Another way that target-specific signalgeneration can be increased is by the use of what has been termed“nested PCR”. In this method, the majority of amplification is carriedout by a second set of primers that flank the labeled primers. This isshown in FIG. 3(C). The labeled primers can be present in reducedamounts, require different annealing conditions or be used in separateshort amplification reactions. This should reduce the involvement of thelabeled primers in amplification of either primer-dimers or non-targetsequences. In this particular instance, it may even be possible tosuccessfully generate target dependent signals with labeled primers ornucleic acid constructs that have some degree of overlap between their3′ ends. Lastly, target independent products should have a differentlength and/or base composition thereby allowing a differentiationbetween target specific double-stranded nucleic acids and inappropriateproducts by their thermal profiles. As described previously, thisprofile can be obtained as part of the process or a separate step may beintroduced to obtain such a profile.

[0216] Although this particular embodiment of the present invention hasbeen described in terms of incorporation of nucleotides, there are alsomeans for extending primers that depend upon the addition ofpolynucleotides rather than individual nucleotides. As such, two ofthese methods, LCR (U.S. Pat. No. 5,494,810) and GAP-LCR (U.S. Pat. No.6,004,286), may also enjoy the benefits of the present invention. Thesemethods depend upon the use of two sets of adjacent oligonucleotideswhere each set is complementary to one particular strand of a targetnucleic acid. In the present invention, a first energy transfer elementwill be in one or more oligonucleotides complementary to one strand anda second energy transfer will be in one or more oligonucleotidescomplementary to the other strand. An illustration of the use of thepresent invention with this method is shown in FIG. 3(D).

[0217] b. Energy Transfer Between a Labeled Primer and Nucleotide(s)

[0218] In another embodiment of the present invention, one or moreprimers or nucleic acid constructs that comprise a first energy transferelement are used in conjunction with at least one nucleotide thatcomprises a second energy transfer element. After target templatedirected addition of nucleotides to the primer or nucleic acidconstruct, energy transfer can then take place by interaction between afirst energy transfer element in one primer or nucleic acid constructand a second energy transfer elements in an incorporated nucleotide. Thelabeled primer or nucleic acid construct and the labeled nucleotide ornucleotides can be on the same strand if only a single primer or nucleicacid construct is used during primer extension events. Linearamplification can also be carried out where the primer or nucleic acidconstruct is used for successive rounds of binding/extension events.

[0219] On the other hand as described previously, the inclusion of oneor more primers or nucleic acid constructs that can use the extendedprimers or extended nucleic acid constructs as templates can allowfurther synthesis. In this case, the second energy transfer elementsthat are introduced by nucleotide incorporation can be in both theextended strand and its complementary copy. FIG. 5 shows potentialamplification products made by various amplification processes thatillustrate this particular embodiment of the present invention. In FIGS.5(A), 5(B) and 5(C), one or more of the primers used for amplificationcontain an energy,transfer element. Although this Figure shows theacceptor element (A) being present in primers and the donor elements (D)being present in the nucleotides incorporated during amplification, theopposite arrangement may also be used. In this particular aspect of thepresent invention, the spacing between the primers can be of any desiredlength that is appropriate for carrying out the amplification.

[0220] As described previously, various methods may be employed toselectively generate signal from only appropriate targets. These caninclude primer design, thermal profiling of double-stranded nucleicacids and nested amplification. This particular embodiment of thepresent invention is also amenable to multiplex formats. For instance,if various primers are used such that more than one extended primerspecies is synthesized, they can be distinguished from each other byusing a common energy transfer donor in the nucleotides and differentenergy transfer acceptors in each of the primers. Each of the individualnucleic acid products can then be identified by the spectralcharacteristics of the acceptor on the primer.

[0221] Previous art has described the dual use of both a primer thatcomprises a first energy transfer element and a dideoxyribonucleotidethat comprises a second energy transfer element (Kwok and Chen, U.S.Pat. No. 5,945,283). The present invention differs from this art inusing nucleotides that-are not strand terminators in the reaction mixthereby a) allowing for the possibility of multiple incorporation eventsand b) allowing sufficient synthesis that the extended strand could beused as a template for synthesis of a complementary nucleic acid ifdesired.

[0222] C. Energy Transfer Between Labeled Nucleotides

[0223] In another embodiment of the present invention, it is disclosedthat signal generation can take place during synthesis with labelednucleotides only. In this particular embodiment, synthesis is carriedout in the presence of at least one nucleotide that comprises a firstenergy transfer element and at least one nucleotide that comprises asecond energy transfer element. The nucleotides that comprise first andsecond energy transfer elements may be the same nucleotide, for instanceby using a mixture of dUTP, where some are labeled with an energytransfer donor and some are labeled with an energy transfer acceptor. Onthe other hand, they may be different nucleotides, for instance by usinga mixture that has dUTP labeled with an energy transfer donor and dCTPlabeled with an energy transfer acceptor.

[0224] As described above, incorporation of nucleotides that comprisefirst and second energy transfer elements can take place during a singleround of strand extension, multiple rounds of extension of one strandfor linear amplification, or by the provision of at least one secondprimer or nucleic acid construct for exponential amplification. FIG.5(D) shows a PCR amplification product where both donors and acceptorshave been incorporated through labeled nucleotides. In the absence ofincorporation, there will be little or no energy transfer between onenucleotide to another. However, once they have been incorporated intonucleic acid strands, they are in position to be able to allow energytransfer to take place. This can be through intrastrand interactions inthe same strand or through interstrand interactons between nucleotideson complementary strands. A particular nucleotide base may consistentirely of labeled nucleotide or there may be a mixture of labeled andunlabeled nucleotides.

[0225] Although methods such as PCR and SDA produce double-strandedamplicons as their major product, there are systems such as NASBA thatalternate between double-stranded DNA and single-stranded RNA forms. Inthese amplification methods, the present invention finds use byproviding either energy transfer labeled deoxyribonucleotides forlabeling the DNA or energy transfer labeled ribonucleotides for labelingRNA products. In the latter case the presence of both donor-labeled andacceptor-labeled ribonucleotides in the RNA strands would allowintrastrand energy transfer. As described previously, various methodsmay be employed to selectively generate signal only from appropriateamplicons. These can include primer design, thermal profiling ofdouble-stranded amplicons and nested amplification. Additionally, sincesignal generation in this particular embodiment of the present inventionis derived from the energy transfer between incorporated nucleotides,the method described by Singer and Haugland (U.S. Pat. No. 6,323,337 B1)can also be used where the primers comprise energy quenchers. Quenchersthat may be used for this purpose can include non-fluorescentderivatives of fluorescein, rhodamine, rhodol or triarlylmethane dyes asdescribed by Singer and Haugland (op. cit.).

[0226] d. Energy Transfer Between a Fluorescent Intercalator and aLabeled Primer or Nucleotide(s)

[0227] The previous embodiments of the present invention have utilizedprimers and nucleotides as energy transfer elements. Another embodimentof the present invention discloses that nucleic acid binding agents canbe used as energy transfer elements after strand extension. It haspreviously been described in U.S. Pat. No. 4,868,103 that energytransfer can be used in a hybridization assay that involves a labeledprobe and an intercalator. In contrast to this art, a labeled primer ornucleic acid construct with a first energy transfer element is extendedto synthesize nucleic acids that can be bound by a nucleic acid bindingagent that comprises a second energy transfer element and issubstantially sequence independent. Binding can take place while theextended strand is still base-paired with its template or afterseparation from the template i.e. the extended strand is indouble-stranded or single-stranded form. The nucleic acid binding agentcan be a protein or a chemical that has a high affinity for nucleicacids. An example of proteins that may find use with the presentinvention may include but not be limited to T4 gene 32 protein, SSBprotein, histones and antibodies. The T4 gene 32 protein and SSB proteinhave affinities for single-stranded nucleic acids and the histones havean affinity for double-stranded nucleic acids. Antibodies specific fornucleic acids and for RNA/DNA hybrids have been described in theliterature (U.S. Pat. No. 6,221,581 and U.S. Pat. No. 6,228,578).Methods for attaching fluorescent labels to proteins have been widelydescribed in the art. An example of a chemical that has a preferentialaffinity for single strand nucleic acids can include but not be limitedto SYBR™ Green II. An example of a chemical that has a preferentialaffinity for double-stranded nucleic acids can include but not belimited to intercalators. Examples of intercalators that may find usewith the present invention can include but not be limited to Acridine,Ethidium Bromide, Ethidium Bromide Homodimers, SYBR™ Green I, TOTO™,YOYO™ BOBO™ and POPO™. The binding agent can comprise a energy transferelement directly or indirectly. Proteins labeled with an energy transferelement would be examples of indirect means. The intercalators listedabove would be examples of direct means.

[0228] Also, energy transfer to or from nucleic acid binding agents canbe carried out by labeled nucleotides instead of labeled primers ifdesired. When the nucleic acid is in double-stranded form, thisembodiment of the present invention can take advantage of the ability ofsome intercalators to have enhanced fluorescence upon binding todouble-stranded nucleic acids. As has been mentioned earlier, thiseffect has been used by itself to monitor real time nucleic acidsynthesis during amplification reactions. However, when used alone, thismethod suffers from the amount of background exhibited by the dye aloneor by dye binding to single-stranded primers. This deficiency may beovercome by the present invention since unbound dye should be unable toundergo an energy transfer interaction with unincorporated labelednucleotides. As such, the present invention should enhance theselectivity of signal generation compared to using a labeled nucleicacid binding agent alone.

[0229] As described previously, various methods may be employed toselectively generate signal from only appropriate target molecules.These can include primer design, thermal profiling of double-strandedamplicons and nested amplification.

[0230] e. Energy Transfer Between a Labeled Probe and Nucleotide(s)

[0231] There may be circumstance where the specificity contributed bynucleic acid probes may desirable. Therefore, another aspect of thepresent invention, discloses novel means of signal generation where atleast one nucleic acid probe that comprises a first energy transferelement is used in conjunction with either nucleotides that comprisesecond energy transfer elements. Previous art has described the use ofan energy transfer labeled primer and an energy transfer labeledsequence specific probe (Wittwer et al. in U.S. Pat. No. 6,174,670). Incontrast to this art, the present method is not constrained to the useof a probe that is in proximity to the primer alone but allows the use aprobe designed to anneal to any location on the nucleic acid strand thatis desirable. In addition, the present invention conveys the ability touse multiple energy transfer probes by using various segments of theextended nucleic acids as probe targets. Thus, when using nucleotidesthat comprise energy transfer elements, signal generation should takeplace after hybridization of labeled probes to the labeled nucleic acidstrand.

[0232] For instance, after strand-extension, the separation or removalof the template strand can allow the binding of a probe to asingle-stranded extended strand and thereby allowing energy transfer totake place between first energy transfer elements in the probe andsecond energy transfer elements that have been incorporated into theextended strand. Energy transfer to or from first elements in the probecan be derived from the segments that are hybridized to the probe, or ifthey are in sufficient proximity, they may be from adjacentsingle-stranded regions. Another illustrative example of the presentinvention makes use of loop-mediated amplification (U.S. patentapplication Ser. No. 09/104,067; European Patent Application PublicationNo. EP 0 971 039 A). One of the structures generated by this system is asingle-stranded loop adjacent to a double stranded stem. Therefore, asdisclosed in the present invention, a probe can be bound to sequences inthe loop region and undergo an energy transfer reaction withincorporated nucleotides.

[0233] In this embodiment of the present invention, specificity isgenerated by two factors. First, the strand extension events should bedependent upon the specificity of the primer binding/extension reactionsthemselves. Secondly, the probes are blocked at their 3′ ends and onlyparticipate by binding to appropriate sequences when these aresynthesized as a result of the primer extension reactions.

[0234] The various embodiments of the present invention that have beendisclosed above can be used in homogeneous assay systems. However, thereare also advantages that are offered by the use of solid supports withthe present invention. Fixation to solid supports can take place priorto initiation of extension reactions, during strand extensions, andafter completion of strand extensions. Examples of solid supports thatmay find use with the present invention can include but not be limitedto beads, tubes, microtiter plates, glass slides, plastic slides,microchip arrays, wells and depressions. Fixation to the support can becarried out either directly or indirectly. As an example of indirectfixation, a capture agent may be attached to the solid support. Thiscapture agent may be a nucleic acid with sequences that arecomplementary to sequences that are present in a primer, a nucleic acidconstruct, an analyte or a copy of an analyte thereby allowing fixationthrough hybridization. Another example of a capture agent could be anantibody that has an affinity for a nucleic acid. (U.S. Pat. No.4,994,373; U.S. Pat. No. 4,894,325; U.S. Pat. No. 5,288,609; U.S. Pat.No. 6,221,581 B1; and U.S. Pat. No. 6,228,578; all of which areincorporated by reference).

[0235] As an example of direct fixation, many of the previousembodiments employ a primer for strand extension. Therefore if desired,these primers could be covalently attached to a solid support prior tocarrying out any extension reactions. In the presence of the appropriatenucleic acids, strand extension can then occur as described previouslythereby resulting in strand extension products that are also directlyattached to the solid support. First and second energy transfer elementscan be in the primers fixed to the solid support and/or they can be innucleotides, probes or nucleic acid binding agents as described in thevarious embodiments disclosed above. An illustrative example of thiswould be the use of amplification on a microarray as described inRabbani et al., U.S. patent application Ser. No. 09/896,897, filed onJun. 30, 2001 (incorporated by reference) with at least one set ofprimers at a locus comprising a first energy transfer element andnucleotides comprising second energy transfer elements. With theappropriate apparatus, each locus on the chip could then be separatelymonitored for the extent of synthesis during the course of theamplification. Another example of direct attachment would be the use ofprimers that comprise ligands and solid supports that comprise theappropriate ligand receptors. An illustrative example of thisarrangement could be the use of poly T primers that have been labeledwith biotin and using beads that are coated with avidin or strepavidinfor fixation. Nucleotides that comprise first energy transfer elementscan be incorporated into a cDNA copy made with the poly T primer and thecomplex of a cDNA bound to its RNA template can bind intercalators thatcomprise second energy transfer elements. In this example, attachment ofthe primers to the beads can take place before, during or after thestrand extensions.

[0236] f. Solid Supports Comprising Energy Transfer Elements

[0237] In addition, a solid support is not relegated to only a passiverole. In another embodiment of the present invention, a solid supportcomprises a first energy transfer element. Fixation of a nucleic acid tothe support can then bring a second energy transfer element intosufficient proximity for a signal to be generated. In this embodiment ofthe present invention, the second energy transfer element can be part ofa nucleotide, primer, probe or nucleic acid binding agent. As anillustrative example, a matrix is made with a selection of nucleic acidprobes on an array. The matrix is then treated such that first energytransfer elements are fixed to the surface as well. In the presence ofappropriate templates or analytes, synthesis of a group of nucleic acidswith second energy transfer elements is then carried out as describedpreviously. Hybridization of the labeled nucleic acids to the arraybrings the second energy elements into proximity with the surface boundfirst elements. Signal should then be generated by energy transfer thatcorresponds to the amount of nucleic acids that are bound to aparticular locus on the array. An illustration of this principle as wellas some of the other embodiments described previously is depicted inFIG. 6. Solid supports with capture elements can also be used in methodswhere no extension reactions are required. For instance, a solidsupport, a capture oligo or an antibody specific for nucleic acids cancomprise a first element. Signal generation can then occur after bindingof an unlabeled analyte in either single stranded or double strandedform to the support where the presence of the second element is dictatedby the presence and amount of the analyte that becomes bound to thesupport. For instance, the second element can be part of the complex inthe form of a probe or a nucleic acid binding agent.

[0238] g. Previous Processes (a Through f) Used for Labeling

[0239] Generation of signal from first and second energy transferelements after target dependent strand synthesis can be used for thepurpose of detecting the presence or amount of a nucleic acid ofinterest in a sample. When signal generation is dependent upon thespecificity of the priming events, the procedures are carried out underconditions where target independent primer extension is minimized or asdescribed previously, methods are included that can distinguish betweentarget derived nucleic acids and spurious strand extension products. Onthe other hand, inclusion of a step that uses the discretionary power ofnucleic acid hybridization can limit or even obviate the need for primerspecificity. For example, an entire library of poly A mRNA sequences canbe converted into a library of cDNA copies by the use of a universalpoly T primer. The library of cDNA strands can then be indiscriminatelyused as templates for second strand synthesis. Inclusion of a promoterinto either the first strand primer (U.S. Pat. No. 5,891,636) or secondstrand primer (Rabbani et al., U.S. patent application Ser. No.09/896,897, filed on Jun. 30, 2001) (both documents incorporated byreference) allows synthesis of multiple RNA copies of each individualoriginal mRNA. When either this product or a cDNA copy is labeled,hybridization with a microarray of nucleic acids can be used todetermine the amounts of any particular species in the original sample.The present invention can be employed with such methods by includingfirst and second energy transfer elements in primers, nucleotides,probes, nucleic acid binding agents or the matrix itself.

[0240] On the other hand, the nucleic acids of interest may be suppliedby the user in known quantity and the embodiments of the variousinvention disclosed above may be used to synthesize labeled probes. Forinstance, a single purified species of a nucleic acid of interest mightbe provided as a template for labeling reactions with one or twoprimers. When a discrete group of varied nucleic acids is desired to belabeled, the primer sets can be expanded accordingly. Labeled probes canthen be created by inclusion of first and second energy transferelements by any of the means disclosed previously. These probes can thenbe used to identify the presence or quantity of unlabeled nucleic acidsin samples in any of a variety of formats that are well known to thoseskilled in the art.

[0241] h. Analytes as Primer Extension Substrates

[0242] It should also be noted that the nucleic acids of interest orcopies of the nucleic acids of interest may also be used directly instrand extension reactions either as substrates for terminal transferaseaddition, ligation or by acting as primers. For example, terminaltransferase can be used for the template independent addition of firstenergy transfer elements onto the 3′ ends of either individual nucleicacids or a library of nucleic acids. Second energy transfer elements canthen be included as part of the terminal addition reaction or they cancomprise primers, probes, nucleic acid binding agents or solid supports.In another illustrative example, restriction digestion of DNA can befollowed by terminal addition of a mixture of nucleotides with first andsecond energy transfer elements. Various individual species of nucleicacids can then be hybridized to various capture sequences on discreteloci of an array to measure the presence or amount of individual labeledsequences.

[0243] It is also a subject of the present invention that analytes orcopies of analytes can be used as primers in template dependent labelingreactions. In this context, incorporation in itself may be used as anassay since template directed synthesis should be dependent upon thepresence of discrete sequences in the analytes that correspond to theircomplements in appropriate hybridized templates. Thus a desired nucleicacid sequence can be specifically labeled in the presence of othernucleic acids that may be present in a mixture. Although at least onesegment of the template is designed to match the desired analytesequence, the segment of the template that is used direct the sequencesadded onto the analyte can be either a natural sequence or an arbitrarysequence.

[0244] As an illustrative example of this method, a library of polyAmRNA can be labeled in the presence of total RNA by using a probe thatcomprises a first segment that comprises Poly T and a second segmentthat can be used as a template. When bound to the poly A sequences ofthe mRNA through the first segment, the 3′ end of the mRNA can beextended using the second segment as a template. The nucleotides thatare incorporated using the second segment as a template can be eitherlabeled or unlabeled. Examples of artificial sequences that may find useas second segments can include primer binding sites, RNA promotersequences. Another illustrative example is a series of probes thatcomprise first segments complementary to the 3′ ends of discretebacterial RNA species. For each particular species-specific segment, adiscrete template sequence can be used. After specific priming by theRNA present in a sample, evaluation can be carried out with an arraythat has capture sequences complementary to the extended sequencesthereby separating out the individual extended RNAs. And lastly, cDNAcopies can be made from a pool of mRNA using standard techniques. EachcDNA that represents a full copy of the original mRNA should have adiscrete 3′ end that represents the 5′ end of the original mRNA.Template/Probes could then be used for each cDNA that is desired to bequantified. In these illustrative examples of using an analyte as aprimer in template dependent extensions, labeling may take place byincorporation of a single labeling species as described in previous artor the methods that have been disclosed above may be employed usingfirst and second energy transfer elements.

[0245] 11. Fragmentation and/or Incorporation of Desirable Nucleic AcidSegments

[0246] a. Template Dependent Addition of Desirable Nucleic AcidSequences to the ends of Analytes

[0247] It is another aspect of the invention to provide novelcompositions and methods for the template dependent addition ofdesirable nucleic acid sequences to analyte or target nucleic acids. Inprior art, poly A sequences have been relied upon as the basis for mostmethods of manipulation of mRNA. Furthermore, the utility of mRNA hasderived from its use as a template to carry out any and all suchmanipulations. For instance, Poly A has been used as a primer bindingsite for making cDNA copies and carrying out linear or exponentialamplification of mRNA. However, as described previously, this feature isnot universally shared among all RNA targets. Furthermore, it is aselective feature for the 3′ end of mRNA. In contrast to this art, thepresent invention overcomes the limited scope of analysis of nucleicacids by viewing and using an analyte or target nucleic acid not as atemplate but as a substrate for strand extension, i.e. as a primer. Assuch, the nucleic acid constructs that are provided for these processesare not used as primers but rather they serve as templates to enableanalyte or target nucleic acids to incorporate any arbitrary sequencethat is desired by the user. Such sequences can comprise promoters,primer binding sites or signal generating moieties.

[0248] In the present invention, methods that may be directed for usewith nucleic acid analytes may also be used with any desirable nucleicacid target as well. Analytes can comprise single desirable sequences orthey may be a library of various sequences. Analyte or target nucleicacids may be comprised of RNA or DNA as well as copies of RNA or DNA.The analyte or targets nucleic acids may be extracted from biologicalsamples or they may have been produced in vitro. They may also haveundergone procedures and processes such as digestion, fragmentation,amplification, extraction and separation.

[0249] The present invention discloses that the ends of nucleic acidscan be hybridized to complementary chimeric nucleic acid constructs(CNACs) that comprise two segments. The first segment comprisesnucleotides or nucleotide analogues that are capable of binding orhybridizing to the 3′ ends of the analytes. The second segment comprisesnucleotides or nucleotide analogues that can be used as a template forextension of the 3′ end of the analyte. In contrast to prior art,methods are disclosed that do not rely upon the presence of a selectedsequence such as a poly A segment at the ends of the anlyte, but ratherthe present invention discloses methods where any and all sequences thatmay be present at the 3′ end of an analyte or library of analyte aresites for binding and template-dependent extension reactions. In thepresent invention, template-dependent strand extension can take placeeither by incorporation of individual nucleotides (polymerization) or byaddition of pre-synthesized oligonucleotides (ligation). It should bepointed out that although the inventions are commonly described in termsof 3′ extension since this is a characteristic of polymerase drivenprocesses, when ligation is used instead, the 5′ end is also a suitablesubstrate for template dependent strand extension. The ability tointroduce arbitary nucleic acid sequences into the 3′ end of a target oranalyte nucleic acid provides a simple and powerful vehicle fortransforming an analyte into a probe or a nucleic acid construct thatcould be used for further manuipulations. A library of nucleic acidswith various sequences can also be converted into a library withuniversal sequences that could be later used for further manipulationdirectly in a controlled and measured manner for signaling purposes,priming events, capture events or amplification events.

[0250] In one particular embodiment of the present invention, a set ofCNACs is used where the first segment comprises all the potentialpermutations of nucleotide sequences. Thus, if the first segments of theset of CNACs comprise 6 variable nucleotides, the first nucleotide (N₁)can be G, A T or C, the second nucleotide (N₂) can be G, A T or C etc.and the set itself will comprise 4⁶ (4,096) different CNACs. In thissense, it has similarity to the use of random primers for synthesis ofnucleic acid copies. However the present invention differs from randompriming in that the CNACs are not extended themselves (i.e., act asprimers) but provide complementary binding to the analytes such that thesecond segment of the CNAC can be used for template dependent extensionof the analyte. For this purpose, it is preferred that the ends of theCNACs be blocked. Thus although the present invention uses randomsequences, the side reaction of random primers using each other asprimers and templates is completely avoided. The present invention alsodiffers in that the binding of random primers to any particular site ofan anlyte allows an extension event. In the present invention, it isonly when the CNAC binds to a complementary sequence at the end of ananalyte that an extension event takes place. Although, there will berandom binding and disassociation of the CNACs at multiple sites on theanalyte strands, this is not a true equilibrium situation since there isactually a dynamic favoring of binding to the ends. For instance,juxtaposition of a 3′ OH in the analyte and a complementary CNAC canbind a polymerase and form a complex that would be more stable than aCNAC bound to an internal site. In addition to providing a longerhalf-life of binding of the CNAC to the terminus by complex formation,the complex generates an even more stable-form by extending the analyte,thereby increasing the number of bases that are complimentarily basepaired. This disequilibrium can be carried out in an isothermalreaction, or if preferred, the reaction temperature can be raised topromote dissociation of CNACs from non-productive binding sites followedby a return to the same reaction temperature to promote another round ofbinding of CNACs to analytes. If desired, these variable conditions canbe recycled multiple times to optimize the amount of analyte ends thatundergo template-dependent addition.

[0251] It is a further objective of the present invention to disclosenovel compositions and methods that utilize CNACs synthesized withuniversal bases i.e., bases that can base pair with more than onecomplementary base. Nucleotides or nucleotide analogues that compriseuniversal bases can contribute stability without adding complexity.Therefore, in this aspect of the present invention, a novel CNAC isdisclosed that comprises two segments as described above, but instead ofusing permutations of nucleotides, universal bases that lack sequencespecificity are used in the first segment. For instance, an example wasgiven above with a set of CNACs that comprised permutations in 6positions thus requiring 4,096 different CNACs. By the use of universalbases, only a single CNAC species is required for providingtemplate-dependent addition of desirable nucleic acid sequences to anyanalytes or set of analytes irrespective of the sequences at their ends.Since universal bases do not always display a complete lack ofdiscrimination and the ability to bind to a particular nucleotide, itwould be possible and even desirable to use a set of CNACs that comprisedifferent universal bases or universal base analogs, or differentmixtures of universal bases and universal base analogs. As describedpreviously, this method can involve a self-selecting process where CNACsundergo a series of binding and dissociation events of the universalbases to random segments of the analytes until the CNAC binds to a 3′end. In this particular embodiment, base pairing at the end is not aproblem since each CNAC possesses universal base pairing capability andproductive extension should be mostly related to the relationshipbetween the 3′ end of the analyte and the second segment of the CNAC.Efficient strand extension can take place where the beginning of thesecond segment of the CNAC is aligned with the 3′ end of the analytesuch that the first base synthesized will be the complement of the firstbase of the second segment. On the other hand, universal bases also havesome capacity for use as templates and as such, hybrids where the 3′ endof the analyte is not perfectly adjacent to the junction between thefirst and second segments of the CNAC should also be able to carry outstrand extension of the analyte.

[0252] It is a further objective of the present invention to disclosenovel compositions and methods where CNACs comprise universal bases incombination with permutations of nucleotides. In this particularembodiment, the CNAC can be considered to comprise three differentsegments wherein a first segment comprises universal bases, a secondsegment represents permuted series of discrete nucleotides at one ormore positions and the third segment comprises a nucleic acid that canbe used as a template for extension of 3′ ends. Thus the presentinvention should be able to enjoy the stability without complexity ofthe universal bases in the first segment in conjunction with selectivityand further stability contributed by specific base pairing by apermutational second segment anchor. Thus, if a universal base used inthe first segment of a CNAC has approximately half of the bindingaffinity as a base pairing between normal nucleotides, a set of CNACsthat comprised 4 variable nucleotide positions and 4 universal baseswould have the same average Tm as random hexamers, but would requireonly 44 or only 256 different CNACs. This is compared to the 4,096different CNACs required with a hexamer permutational first segment.Similarly a CNAC with 4 universal bases and 6 variable positions wouldcomprise 4,096 different CNACs but would have binding propertiesanalagous to CNACs with random octamer first segments that would require4⁸ permutations (i.e. 65,536 different CNACs).

[0253] Therefore, one would cover all possible permutationalcombinations that could exist in the terminal end of any analyteregardless of its derivation, while at the same time enjoying reasonablyhigh binding efficiency and capabilities because the universal sequencesin the first segment provide the additional binding stability withoutimposing any further specificity. Thus for the same number of CNACmolecules in a reaction mixture, there would effectively be a 16 timeshigher concentration of CNACs that could bind to a particular 3′ end ofan analyte in the examples cited above. This should provide superiorkinetics and efficiency compared to CNACs with only permutationalsegments.

[0254] Universal bases, i.e., bases that can base pair with more thanone complementary base, were first used in oligonucleotides to maintainstable hybridization with target nucleic acids that had ambiguity in theidentity of their nucleotide sequence. A well-known example of this isthe substitution of inosine in PCR primes (Liu and Nichols, (1994)Biotechniques 16; 24-26). Inosine has the property of being able to basepair efficiently with either G, A, T or C in a complementary strand(Kawase et al., 1986, Nucl. Acids Res. 19; 7727-7736). The meltingtemperature is less than a normal base pairing but still higher than amismatch. When used as a template, inosine is recognized as if it waseffectively G and a C is preferentially incorporated into thecomplementary copy. Other analogs of nucleotides that can act asuniversal bases have also been described. For instance,5-nitroindolenine and 3-nitopyrrole analogues have also been describedas universal bases (Loakes and Brown, 1994, Nucl. Acids Res. 22;4039-4043, Nichols et al., 1994, Nature 369; 492-493 both of which areincorporated by reference). The use of these and other universal basesare reviewed by Loakes (2001) in Nucl. Acids Res. 29; 2437-2447(incorporated by reference). The ability of universal bases to addstability without adding to the complexity of primers has been describedby Ball et al., (1998, Nucl. Acids Res. 26; 5225-5227, incorporated byreference) where the addition of 5-nitroindolenine residues at the 5′end, improved the specificity and signal intensity of octamer primersused for cycle sequencing. Thus, these and other universal bases may allfind use in the present invention.

[0255] As described above, the present invention allows any nucleic acidor nucleic acid fragment to be used for template-dependent extension andobviates dependency upon poly A tails. The desirable nucleic acid (ornucleic acid of interest) that is incorporated into an analyte strandcan transform any nucleic acid or nucleic acid fragment into a form thatprovides a primer binding sequence that can carry out functionspreviously enjoyed by polyadenylated nucleic acids. Linear amplificationcan be carried out by incorporating a promoter as the desirable nucleicacid (or nucleic acid of interest) in a CNAC and exponentialamplification can be carried out with desirable primer binding nucleicacid sequences using any of the methods previously described for poly Atargets. Additionally, it is contemplated that template-dependentincorporation of a nucleic acid into an analyte also presents theopportunity to directly label the analyte or analytes by using a labelednucleotide or oligonucleotide in the incorporation step.

[0256] Research studies have had a focus on poly A mRNAs due to itsaccessability and convenience as a substrate. The present inventionallows non-polyadenylated nucleic acids to be manipulated with the sameease of use previously accorded to poly A mRNA. Thus, the presentinvention can be used with DNA, hnRNA, snRNA, tRNA, rRNA, bacterial mRNAor any RNA lacking a poly A sequence. Even poly A mRNA may find use withthe present invention. The reliance upon the 3′ poly A tail has led to abias towards the information contained in this end. In most methods ofprior art, sequences at the other end of mRNA were still dependent uponthe efficiency with which a priming event at the 3′ took place.Accordingly, any interruptions in the copying process or a scissionbetween the 5′ end and the poly A end reduced the amount of 5′ sequencesthat were available for study or manipulation. Thus, even a single nickin a large mRNA molecules eliminated the use of the 5′ end of themolecule and numerous reports and even commercial products are dedicatedtowards the preservation of the continuity between the 5′ and 3′ ends ofmRNA during its isolation. Since the present invention discloses methodsthat are independent of poly A, fragments of poly A RNA that have becomeseparated from the poly A region remain available for use and study.

[0257] In fact, such a fragmentation process can be advantageous sinceall segments of the poly mRNA can be independently and efficiently usedwith no bias derived from their relationship to the 3′ end. Thisfragmentation will be especially useful for hnRNA which has remained anunderutilized area of research. This neglect has stemmed from twocharacteristics of hnRNA: the lack of poly A as a handle and the verylarge average size. Although the introns that are present in hnRNa lackcoding sequences for the final gene product, there are likely to be alarge number of sequences that do not appear in the final product thatare important in control, regulation and interaction with other genesand gene products. The present invention will allow the sequencespresent in hnRNA to be as completely accessible as the polyA mRNAsequences had been previously.

[0258] Although many of the embodiments of the present invention aredescribed in terms of RNA analytes, it should be pointed out that manyof these processes can easily be applied to DNA fragments as well.Methods that can be used for the fragmentation processes described abovecan be physical or enzymatic. Physical means can encompass any chemicalprocess as well as mechanical shearing and sonication. Enzymaticprocesses for fragmentation that may find use with the present inventioncan include but not be limited to endoucleases such as S1 nuclease, mungbean nuclease, RNase, DNase and restriction enzymes. It is a furtherpoint of the present invention that analytes can be treated withphosphatases if required, to provide an extendable 3′ end. The CNAC ofthe present invention can comprise DNA, RNA or any combination thereofand the nucleotides may be modified or unmodified as desired. The CNACsmay comprise standard nucleotides or they may comprise nucleotideanalogs, sugar analogs and phophate analogs. Examples of each of theseare peptide nucleic acids (PNAs), arabinosides and phosphorothioatelinkages.

[0259] b. CNAC for Site Specific Fragmentation

[0260] The utility of universal bases to providing stability withoutadding complexity finds application in other processes as well. Anotheraspect of the present invention discloses compositions and methods forcontrolled fragmentation of an analyte or library of analytes. A novelCNAC is disclosed that comprises two segments, a first segment thatcomprises universal nucleotides to provide non-specific binding and asecond segment with a discrete selected sequence that will generate acomplex that provides endonucleolytic digestion. Under appropriatehybridization conditions, the CNAC will create an endonucleasesusceptible site at each location in the analyte that is sufficientlycomplementary to the second segment of the CNAC. The size and nature ofthe selected sequence will determine the average spacing betweenendonuclease sites and therefore the particular average size offragments. For example, a CNAC that comprises a second segment with 4 ormore deoxyribonucleotides should form a complex that is a substrate forRNase H. This should lead to a scission at each site in the analyte thatis complementary to the second segment. On average, a given 4 basesequence should appear about every 250 bases. A smaller sizedistribution can also be obtained by the use of more than one CNACthereby increasing the number of potential digestion sites. Ifpreferred, a larger second segment can be used andhybridization/digestion conditions applied such that the complex isformed at more infrequent intervals and hence a larger averagedistribution in fragment sizes. Specificity may also be increased by theaddition of discrete bases in either the first or third segments andusing conditions such that stable hybrids are only formed with stabilitygenerated by proper base pairing of these bases as well.

[0261] The same method can also be applied to digestion ofsingle-stranded DNA. The second segment of a CNAC can be designed with arecognition site for a restriction enzyme. Since most restriction sitesare only 4 to 6 bases, the presence of the universal bases in the CNACshould provide a much more stable hybrid than using a 4 to 6 basesegment alone. Although in this particular embodiment of the presentinvention, the second segment is used for fragmentation, it may also beused as a template for strand extension for incorporation of a desirablenucleic acid sequence into a fragmented abalyte after endonucleolyticdigestion. As described previously, this can provide a means fortemplate-dependnent incorporation of a labeled nucleotide oroligonucleotide to label the analyte fragments at their terminus.

[0262] If preferred, the CNAC disclosed above can further comprise athird segment. For instance, the third segment can comprise another setof universal bases flanking the other side of the discrete bases in thesecond segment. This CNAC could be represented by the formula“U_(n)-D_(p)-U_(q)” where the “n” represents the number of universalbases in the first segment, “p” represents the number of discrete basesin the second segment and “q’ represents the number of universal basesin the third segment. The additional third segment can provideadditional stability or it may make the hybridized second segment a moreefficient enzyme substrate for endonucleolytic digestion. Alternatively,the first and second are as described above and the third segment is adiscrete nucleic acid sequence that provides a template forincorporation of one or more labels or a desirable nucleic acid sequenceas described previously. Since the universal bases allow forindiscriminate binding, the reactions can take place under conditionswhere only hybridization events that include proper alignment with thediscrete bases in a CNAC form stable hybrids between the CNAC and theanalyte. Alternatively, thermocycling can be carried out to dissociateCNACs that are non-productively bound and allow additional bindingevents that lead to site-specific fragmentation until substantially allof the desired sites on the analyte have been digested.

[0263] C. CNA Cs for Digestion/Extension

[0264] In another aspect of the present invention, novel CNACs aredisclosed that comprise at least two segments where the first segment iscomplementary to a first analyte nucleic acid sequence and the secondsegment is complementary to a second analyte nucleic acid sequence. TheCNAC is designed such that after mixing it with an analyte nucleic acid,hybridization of a first segment to a first analyte nucleic acidsequence forms a first complex that is resistant to a particularendonuclease, while hybridization of a second segment to a secondanalyte nucleic acid sequence forms a second complex that is a substratefor the endonuclease. Furthermore, the second complex is capable ofasymmetric cleavage such that only the analyte strand is subject tonicking or removal of nucleotides by the endonuclease. This treatmentgenerates a new 3′ end in the analyte strand that can then be used forthe template dependent addition of nucleotides or oligonucleotides tothe analyte strand.

[0265] The CNAC may further comprise a third segment that may or may notbe complementary to a third analyte nucleic acid sequence. The thirdsegment of a CNAC is distinguished from a second segment in that a thirdsegment does not generate a third complex that is sensitive toendonucleolytic digestion. When the third segment is not complementaryto the third analyte nucleic acid sequence, a third complex is neverformed. On the other hand, when the third segment is complementary to athird analyte nucleic acid sequence, a third complex is formed, butendonuclease resistance is endowed by any of the means that can beemployed to render a first complex resistant. After endonucleasedigestion, the sequences in the second and third segments may act astemplates for strand extension from a 3′ end that has been generated byaction of the endonuclease. The strand extension may be carried out by atemplate-dependent polymerizing enzyme (DNA polymerase or reversetranscriptase), or a template dependent ligation enzyme (DNA ligase).Fragments generated by endonuclease digestion may be further besubjected to kinase or phosphatase treatment, in order to add or removephosphate groups at the 3′ or 5′ end as may be desired.

[0266] Analytes that may find use in the present invention can be eitherbe DNA or RNA depending upon the nature of the CNAC and theendonuclease. Sequences in the analytes that may be used in the presentinvention may be discrete individual sequences, consensus sequences, orgeneric sequences that are present in all or most of a library ofanalytes. Examples of RNA that may find use with the present inventioncan include but not be limited to hnRNA, rRNA, mRNA, tRNA, or snRNA.Examples of DNA that may find use with the present invention can includebut not be limited to chromosomal, single-stranded, plasmid, viral,bacterial DNA. Digestion of the second complex can be carried out byendonucleases such as RNase H and restriction enzymes. Prior tohybridization with the CNAC, the target nucleic acid or analyte nucleicacid may also have undergone pre-treatments including, digestion,fragmentation, extraction and separation. These fragmentationpre-treatments can include physical means, such as shearing, sonicationor chemical treatment. Pre-treatments may also include endonuclease orexonuclease digestions. Examples of endonucleases that might find use inthe present invention for pre-treatment can include but not be limitedto Si nuclease, mung bean nuclease, restriction enzymes, DNAse,ribonuclease H and other RNases.

[0267] The various segments of chimeric nucleic acid construct polymermay be comprised of the same or different backbones. For example, afirst segment of a CNAC can comprise oligo-ribonucleotides and thesecond segment can comprise oligo-deoxyribonucleotides. Generally, thesugar-phosphate backbone may comprise a natural element, such asphosphate, ribose, or deoxyribose, or it may comprise analogs ofphosphates such as phosphorothioates, or analogs of sugars such asarabinosides. If desired, the 3′ or 5′ end of a CNAC may be blocked toprevent it from acting as a primer or from participating in ligation.The segments of a CNAC may further be comprised of a synthetic backbone,such as a polypeptide. Any synthetic polymer can be used as backbone aslong as bases can be added in the proper orientation so that basepairing can take place. A prominent example of such a synthetic polymerthat has this capability and usefulness is a peptide nucleic acid (PNA).The bases may be comprised of natural purine and pyrimidine bases aswell as modified versions thereof. The bases may also comprise analogsof natural bases. For instance, the universal bases discussed previouslymay also find use in this embodiment of the present invention. Differentsegments of a CNAC may comprise the same or different backbones andcomprise any base structures or elements, depending on the desirablefunction. Thus, one can construct a desired CNAC from the variouscomponents and elements provided above. The particular choice ofcomponents will depend upon the nature of the analyte and theendonuclease to be used.

[0268] For example, if the analyte is an RNA molecule, RNase H can beused as the endonuclease when the backbone of the first segmentcomprises an oligo-ribonucleotide and the backbone of the second segmentcomprises oligo-deoxyribonucleotides. Consequently, hybridization of thefirst segment to an RNA analyte creates a double-stranded RNA firstcomplex that is resistant to ribonucleaseH and an RNA-DNA second complexwhich is a substrate for Rnase H activity. Treatment with RNase H wouldasymmetrically cleave all or some of the portion of the RNA analyteinvolved in the second complex but leave the RNA-RNA hybrid of the firstcomplex and the second segment of the CNAC intact. As described above, aCNAC may also comprise a third segment. In the example above, if thethird segment is complementary to the RNA analyte, the third segment mayalso comprise an oligo-ribonucleotide such that hybridization to theanalyte forms an RNA-RNA hybrid that is resistant to the action of RNaseH. Alternatively as described above, the third segment is notcomplementary to the RNA analyte and no hybrid is formed.

[0269] The choice of the particular endonuclease used to carry out thisaspect of the present invention depends upon a number of factors. Theprimary factor is the nature of the analyte since the endonuclease mustbe able to utilize the analyte as a substrate for nicking or removal ofnucleotides. Secondly, the endonuclease must allow circumstances wheresuch nicking or removal is substantially asymmetric and takes place inthe analyte strand. Thirdly, the endonuclease must allow circumstanceswhere a first or third complex can remain substantially resistant to theaction of the endonuclease. Lastly, the endonuclease must havesufficient specificity that it acts only upon the portion of an analytethat participates in formation of a second complex with the CNAC. It canbe seen that the illustrative example with RNase H described abovefulfills all of these criteria.

[0270] Another illustrative example would be to utilize an endonucleasethat intrinsically provides an asymmetric cleavage. For example,digestion of double stranded DNA with the restriction enzyme N.BstNB Iresults in a nick in only one strand 4 bases downstream from therecognition sequence 5′ GAGTC 3′. Thus, one could design the secondsegment of a chimeric nucleic acid construct with anoligo-deoxyribonucleotide sequence that is complementary to thissequence and a first segment that is complementary to sequences that areadjacent to the binding site for the second segment. In such a manner,when a second complex is formed by hybridizing the CNAC to the analyte,the double-stranded DNA is a substrate for this specific restrictionenzyme and only the analyte sequence will undergo cleavage. As describedpreviously, the CNAC can comprise a third segment that can serve as atemplate for introduction of a novel nucleic acid sequence by additionto the 3′ end of the nick created by the endonuclease digestion. Thisexample also serves as an illustration that a CNAC can still beconsidered “chimeric” even when it is a chemically homogeneous molecule.For instance, the CNAC above can be synthesized with three segments thatcomprise only oligo-deoxyribonucleic acids. In the present invention,this would still be a chimeric molecule since each segment has adifferent functional property, i.e., the first segment providescomplementary base pairing and stability; the second segment providesfor endonuclease susceptibility and the third segment provides atemplate for strand extension. This method may also be combined withother embodiments of the present invention that have been disclosedpreviously. For instance, a CNAC with two segments can compriseuniversal bases with specific nucleotides only in the sites that arerequired for recognition and digestion by the asymmetric endonucleasedescribed above.

[0271] Another illustrative example of how this aspect of the presentinvention could be carried out would be by the use of an artificial orsynthetic second segment where the constituents are modified or compriseanalogs. Any such modification or analog may be used for this purpose aslong as a) they allow hybridization to occur between the second segmentand the analyte b) hybridization with the analyte forms a complex thatis susceptible to endonuclease digestion and c) the second segmentremains substantially resistant to the action of the endonuclease. Forexample, a second segment could comprise phosphorothioate linkagesbetween bases. It has previously been shown that when a restrictionenzyme site in a double stranded molecule comprises an unmodifiedsegment and a phosphorothioate segment, only the unmodified segmentundergoes a cleavage event (U.S. Pat. No. 5,270,184 and U.S. Pat. No.5,455,166; incorporated herein by reference). Thus a CNAC with one ormore phosphorothioate linkages in a restriction enzyme sequence in asecond segment can be hybridized to a complementary segment of ananalyte and only the analyte strand should be subject to endonucleasedigestion.

[0272] Generally, the third segment may contain any arbitrarysequence-segment, either related or non-related to a target nucleicacid. The third segment provides a template upon which the cleavedtarget nucleic acid or the analyte can act as a primer and thereby allowthe introduction of any desirable nucleic acid sequence into an analyte.Through such a template dependent sequence introduction to an analyte, asignaling moiety or other elements such as primer binding sequences canbe introduced directly to an analyte. Furthermore, through such amethod, universal sequences could be introduced to an analyte nucleicacid that could act at a later stage as a template for the introductionof a universal primer or primer directed promoter system to preparecopies of the analytes as described in U.S. Pat. No. 5,891,636 andRabbani et al., in U.S. patent application Ser. No. 09/896,897; both ofwhich are incorporated by reference. The fact that nucleic acidfragments can be converted to such a construct through such a methodcould provide for an even amplification of nucleic acid librarieswithout prejudice to 3′ end sequences. Further, such a sequence orsequences could be used for priming or capturing events directly orafter amplification. Optionally, if endonuclease cleavage does not leavefree 3′—OH in the remaining analyte, then the remaining analyte could betreated with phosphatase so that a 3′—OH is generated which canfacilitate a priming event. Washing, melting or separation steps can beemployed when and where desirable. Generally, with a chimeric nucleicacid construct with three sequence segments, one can introduce at willdesired nucleic acid sequences at any location into an analyte nucleicacid sequence, including any possible internal sequence sites.

[0273] The various teachings in the present invention allowsintroduction of desirable specific sequences in a template directedmanner into an analyte and thus empowering the analyte or set ofanalytes with diverse properties and capabilities including: acting as aprobe; as a template; as a primer. The CNAC and/or an analyte labeled bymeans of a CNAC can be directly or indirectly immobilized onto a solidsupport which may include: tubes, cuvettes, plates, microtiter wells,beads, magnetic beads, and chips. Methods and compositions for carryingout this particular embodiment are described in U.S. Pat. No. 4,994,373;U.S. Pat. No. 4,894,325; U.S. Pat. No. 5,288,609; and U.S. Pat. No.6,221,581 B1; U.S. Pat. No. 5,578,832; and U.S. Pat. No. 5,849,480 (allof which are incorporated by reference). This immobilization can takeplace either before or after strand extension and labeling of ananalyte. For instance, such capabilities could be used in nucleic acidarray analysis, in which instead of probing the analyte, the analyteacts as a primer on a matrix comprising an array of CNACs that canprovide templates for strand extension of diverse analytes. Dependingupon the particular embodiment of the present invention, hybridizedanalytes may be extended directly or undergo an endonuclease step priorto extension. One or more labels or signaling moieties could beincorporated directly or indirectly with such an array to indicate aspecific hybridization of analytes to a site on the array.

[0274] d. CNAC for Partial Removal of Homo Polymeric Sequences

[0275] Another aspect of the present invention discloses novelcompositions and methods for the partial removal of a homopolymersequence. Homopolymeric sequences are naturally present in poly Amessenger RNA and are artificially present in many methods used forcloning. An example of the latter is poly C and poly G tailing ofdouble-stranded cDNA molecules (Okayama and Berg, 1982 Mol. Cell. Biol.2;161). Although the presence of these homopolymeric tracts providebeneficial effects for universal primer binding and cloning, only asmall segment is usually necessary and the presence of large segmentsmay actually be problematic. For instance, in a transcription templatemade from a cDNA cop of mRNA, long homopolymeric segments may inducepremature terminations.

[0276] As such, the present invention discloses a CNAC that comprisestwo segments. The first segment is complementary to a chosenhomopolymeric sequence and is designed such that a complex formedbetween the homopolymeric sequence and the first segment forms a firstcomplex that is resistant to the action of a particular endonuclease.The second segment also comprises a sequence complementary to thehomopolymeric sequence, but forms a second complex that allowsendonuclease digestion of the homopolymer. Thus although each of thesegments comprise sequences complementary to the same target sequence,they differ in the properties they will confer after hybridization.

[0277] For instance, a CNAC that comprises a first segment made of rUand a second segment comprised of dT can hybridize to any segment of apolyA tail of mRNA. Digestion with RNase H will only eliminate poly Asegments hybridized to the second segment. The CNAC can be recycledmultiple times either by using thermal cycling or a temperature wherethe hybridization through a first segment or a segment alone isinsufficient for stable hybridization. For instance, a CNAC that iscomprised of 10 rU and 10 dT bases would be able to efficientlyhybridize to a 20 base poly A segment at 37° C. Elimination of rA basesin this segment through RNAse H activity should destabilize the CNAC,enabling it to bind to a new segment. This process should continue untilthe mRNA molecules have has less than 20 rA bases left at their 3′ ends.The remaining small poly A segment can then be used as a primer bindingsite by using appropriate hybridization conditions. If the CNAC or a DNAprimer containing olgo T is used for this purpose, it is preferred thatthe RNase H activity used for the digestion be eliminated prior topriming.

[0278] The CNAC described above for generating resistant and sensitivecomplexes is meant only to exemplify the present invention and othersizes may be used for first and second segments. For instance, adeoxyribonucleotide segment of four bases have been shown to besufficient for forming complexes that are substrates for Rnase Hactivity. The size of the segments of the CNAC should be designed suchthat there is efficient complex formation prior to endonucleasedigestion and a sufficient portion of the homopolymeric target remainsintact under the condition used for endonuclease digestion.

[0279] Furthermore, the CNACs of the present invention can comprise athird segment that may or may not be complementary to the homopolymerictarget sequence. As described previously, if the third segment iscomplementary, the nature of the endonuclease and third segment is suchthat a third complex remains resistant to digestion by the endonuclease.The third segment can be homopolymeric or heteropolymeric depending uponits intended purpose. The nucleotides in the various segments of theCNAC may be comprised of natural bases or analogs thereof, universalbases or combination thereof that may provide either a weaker orstrengthened hybrid formation with a desired sequence. For instance, theuse of universal bases in a third segment can allow synthesis of acomplementary segment that has a weaker than normal binding. Thus, ifthe new segment on the analyte is desired to be used as a primer bindingsite, a primer with normal base that were complementary to the primerbinding site would have a competitive edge over re-annealing by theuniversal bases in the CNAC.

[0280] It will be readily appreciated by those skilled in the art thatany of the compositions, solid supports, reagents, dyes, primers,nucleic acid constructs, and the like, can be formulated as kits, whichcan be employed for carrying out any of the processes described orclaimed herein, and variations of such processes. For example, kits canbe formulated as protein or nucleic acid labeling kits, nucleic acidprocessing kits, kits for incorporating desired nucleic acid sequences,amplification kits for amplifying targets, analytes and even a libraryof analytes. Post-synthetic and real time amplification kits can also beformulated from the compositions, solid supports, reagents, dyes,primers, nucleic acid constructs, and the like.

[0281] The following examples are offered by way of illustration and notby way of limitation to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 Preparation of Cy 3Labeling Reagent

[0282] (a) Preparation of Compound I (2,3,3-Trimethylindolinium5-Sulfone)

[0283] P-Hydrazinobenzenesulfonic acid (250 g) was mixed with glacialacetic acid (750 ml) and 3-methyl-2-butanone (420 ml) and heated atreflux for 3 hr. The solution was poured into a 2 L beaker and allowedto cool overnight. The resultant suspension was filtered, washed withacetic acid and lyophylized to remove residual acetic acid. Theresultant solid was dissolved in methanol (1.5 L) and a saturatedsolution of potassium hydroxide in 2-propanol (900 ml) was slowly added.The color of the solution turned progressively lighter as the potassiumsalt of 2,3,3-trimethylindolinium 5-sulfone precipitated. Theprecipitate was filtered by suction, washed with 2-propanol andlyophilized to dryness to give 238 g of Compound I.

[0284] (b) Preparation of Compound II

[0285] (1-Ethyl-2,3,3-Trimethylindolenineninium 5-Sulfone)

[0286] A portion (78 g) of Compound I synthesized in step (a) wassuspended in 1,2-dichlorobenzene (700 ml). Ethyl iodide (250 ml) wasadded and the mixture was heated at 90-100° C. for 12 hr while stirring.The mixture was poured into 3 L of a 1:1 mixture of ethylacetate/etherand stirred for 2 hours. The resulting precipitate was filtered, washedwith a 1:1 mixture of ethylacetate/ether and air-dried to give 68 g ofproduct, Compound II.

[0287] (c) Preparation of Compound III (6-Bromohexanoyl Allyl Amide)

[0288] 6-Bromohexanoic acid (20 g) and N-hydroxysuccinimide (15 g) weredissolved in 200 ml of anhydrous dimethylformamide (DMF).Dicyclohexylcarbiimide (22 g) in anhydrous DMF (50 ml) was added and themixture was left at room temperature overnight. The precipitated ureawas removed by filtration and the DMF solution containing the product,N-hydroxysuccinimide-6-bromohexanoate, was cooled to −10 to −20° C. Anequimolar amount of allylamine in H₂O (11 ml) was first brought to pH8-9 with glacial acetic-acid and then added slowly with stirring to theactive ester. Solid sodium bicarbonate (10 g) was added slowly to avoidexcessive foaming and the mixture was left without covering until thetemperature was raised to −10° C. in two hr. The mixture was poured intoH₂O (1L) and the product was extracted twice with chloroform (300 ml).The extracts were washed once with 1 N HCl in H₂O, once with 5% NaHCO₃(300 ml) and three times with 10% NaCl in water. The chloroform phasewas dried by addition of solid MgSO₄ and leaving it overnight understirring. The chloroform was removed by evaporation under vacuum leavinga liquid that was used without any further purification for the nextstep.

[0289] (d) Preparation of Compound IV (Addition of Linker Arm toCompound III)

[0290] Compound I (11 g) from step (a) and Compound III (15 g) from step(c) were dissolved together in 1,2-dichlorobenzene (100 ml) and heatedat 110° C. for 12 hours while stirring under argon. The mixture wasslowly poured into ethylacetate a 1:1 mixture of ethylacetate/ether (700ml) and after 30 minutes the solid precipitate was filtered, washed witha 1:1 mixture of ethylacetate/ether, air-dried and set aside. A glassysolid that was formed at the bottom of the flask was crushed in amortar, triturated with a 1:1 mixture of ethylacetate/ether, filtered,washed with 2-propanol, dried in vacuum and combined with theprecipitate from above to give Compound IV which was used without anyfurther purification.

[0291] (e) Synthesis of Cy 3 Labeling Reagent (Compound V)

[0292] A portion of Compound II (12 g) from step (b) andN,N′-diphenylformamidine (10 g) in acetic acid (60 ml) were heated at100-110° C. for 90 min with stirring. During the reaction the absorptionat 286 nm and 415 nm was measured. The ratio of 415/286 increased duringthe first 60 minutes then remained constant at 2.2 for the next 20minutes. After 90 minutes, the hot mixture was poured slowly into 700 mlof a 1:1 mixture of ethylacetate/ether. The resultant solid precipitatewas collected with a pressure filter funnel, washed with 1:1 mixture ofethylacetate/ether and dried by passing argon through the cake. Theprecipitate was collected from the pressure filter funnel and slowlyadded to a mixture of 6.5 g of Compound IV from step (d), 50 ml ofpyridine and 50 ml of acetic anhydride. The progress of the reaction wasmonitored by the decrease of absorbance at 385 nm and an increase inabsorbance at 550 nm. The reaction was carried out overnight understirring at room temperature. The absorbance at 550 nm increased withtime followed by a drop in absorbance as the product precipitated out ofsolution. At the end of the reaction, the brown precipitate wascollected and put aside. The liquid portion was treated by the additionof a seven-fold volume of ethylacetate. The precipitate that formed wascollected and combined with the first precipitate. Since pyridine wouldinterfere with a later palladium catalyzed step, any remaining pyridinewas removed by dissolving the combined precipitate in 100 ml of 0.5MTriethylammonium carbonate, pH 8.0 (TEAC). The TEAC was then removed byevaporation under vacuum leaving a solid pellet. This product (CompoundV) was then dissolved in H₂O and kept at −70° C. until ready to be used.

EXAMPLE 2 Preparation of Cy 5 Labeling Reagent (Compound VI)

[0293] Compound II (8 g) from step (b) of Example 1 and malonyl aldehydedianil hydrochloride (10 g) were dissolved in 100 ml of a 1:1 mixture ofglacial acetic acid and acetic anhydride followed by heating at 110° C.for two hours. The mixture was slowly poured into 500 ml of a 1:1mixture of ethylacetate/ether and the precipitate was filtered, washedwith a 1:1 mixture of ethylacetate/ether and dried by argon as above.The precipitate was then slowly added to a mixture of 12, g of CompoundIV dissolved in 150 ml of a 1:1 mixtutre of pyridine/acetic anhydridewhile stirring. The mixture was transferred to an oil bath maintained at90-100° C. for 30 minutes while continuing to stir. If desired, thisstep could have been extended up to 90 minutes. The reaction mixture wasthen cooled to room temperature and the precipitate was processedfurther as described previously for the Cy 3 labeling reagent in Example1.

EXAMPLE 3 Attachment of Cy 3 (Compound V) to dUTP

[0294] Mercurated dUTP (30 umoles) prepared as described in U.S. Pat.No. 5,449,767 was dissolved in 1 ml of 1M Lithium acetate and the Cy 3labeling reagent (60 umol, 0.6 ml) prepared in Example 1 (Compound V)was added with stirring. Potassium tetrachloropaladate (30 umol in 0.5ml H₂O) was added under argon. The reaction was monitored by HPLC andwas complete after 1 hr at 40° C. Overnight incubation did not increasethe yields. Four volumes of acetone were added to the reaction mixtureand left overnight at −20° C. The next day, the precipitate wascollected by centrifugation.

[0295] The pellet was dissolved in 0.1M Lithium acetate (pH 4) andloaded onto a DEAE Sephadex A₂₅ column. The column was developed bypassing through a linear gradient of 0.1-0.7 M LiCl in 0.1M Lithiumacetate. The fractions were examined by HPLC and the fractions whichcontained a single late peak were collected and set aside. Another groupof fractions exhibited two peaks: the late peak described above and anearlier peak. These fractions were combined, adjusted to 0.1 M LiCl,reloaded onto a DEAE Sephadex A₂₅ column and refractionated as above.Again the fractions containing a single late peak were collected and setaside. Although it was not done in this example, the fractions thatcontained two peaks after the second chromatography could have beencombined and put onto the column another time to increase the yield ofthe single peak product. The fractions that had exhibited a single latepeak by HPLC were combined together and the H₂O was removed byevaporation in vacuum. The last traces of H₂O were removed byresuspension of the semi-solid residue in 50 ml of 100% ethanol followedby evaporation. The alcohol step was repeated once more. The residue wasresuspended in 30 ml of ethanol and 1 ml of 3M lithium acetate wasadded. The solution was mixed well and left overnight at −20° C. tofacilitate complete precipitation of the Triphosphate. The precipitatewas collected by centrifugation, redissolved in H₂O and partiallylyophilized to remove remnants of the ethanol. The amount of product wasmeasured by absorbance at 550 nm and a molar extinction value of150,000. The solution was then readjusted to a stock concentration of 10mM and stored at −70° C.

[0296] Although the procedure above describes the preparation of Cy3labeled dUTP, the same steps could be carried out for the preparation ofCy 5 labeled dUTP by the substitution of the Cy5 labeling reagent(Compound VI from Example 2) instead of theCy3 labeling reagent(Compound V from Example 1) used in the example above.

EXAMPLE 4 Preparation of a Labeled Nucleotide with a Rigid Arm Linkerand an Aphenylic TAMRA Analogue

[0297] (a) Preparation of Compound VII(3,6-Bis-(Dimethylamino)-Xanthene-9-Propionic Acid)

[0298] 3-(Dimethylamino)phenol (5.8 g) was mixed with succinic anhydride(2.1 g) and heated at 120° C. for 90 minutes with stirring under argon;The mixture was cooled, H₂O (80 ml) was added and the mixture was heatedat reflux for 10 minutes. The water phase was discarded, leaving behinda dark brown gummy material. This substance was dissolved by theaddition of H₂O followed by an adjustment to pH 10 with 1M NaOH whilestirring. The pH of the clear solution was then brought down to 2 by theaddition of 1M HCl. The dye was salted out by the addition of NaCl to afinal concentration of 2.5. M. The precipitate was filtered, washed withNaCl (2.5 M) and dried by lyophilization to give 1.2 g of Compound VII.The fluorescence spectrum of Compound VII is shown in FIG. 7.

[0299] (b) Preparation of Compound VIII (Active Ester of Compound VII)

[0300] Compound VII from step (a) was dissolved in chloroform (200 ml)and N-hydroxysuccinimide (1.5 g) was added with stirring. After theN-hydroxysuccinimide went into solution,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (2 g) was added and themixture was stirred overnight in the dark. The mixture was extractedwith H₂O (80 mL) and the chloroform phase containing Compound VIII wasdried with anhydrous magnesium sulfate and stored at −20° C.

[0301] (c) Preparation of Compound IX (Free Acid Form ofGlycerlyglycine)

[0302] 13 g of glycylglycine was suspended in a mixture of an equimolaramount of triethylamine in 300 ml of anhydrous methanol and mixed with a1.5 molar excess of methyltrifluoro acetic ester. The suspension wasrefluxed until a homogeneous solution was achieved. The methanol wasremoved by rotary evaporation and the residue was suspended in 100 ml ofH₂O. The pH was then adjusted to 10.0 to allow thetrifluoroglycerylglycine to go into solution. The pH was then broughtdown to 1-2 with Hydrochloric acid whereupon the free acid form of thetrifluoroglycerylglycine precipitated out of solution. This mixture wasleft overnight at 4° C. to allow complete precipitation of the product.The next day, the precipitate (Compound IX) was collected by filtrationand then dried

[0303] (d) Preparation of Compound X (NHS Ester of Glycerylglycine)

[0304] 15 g of Compound IX from step (c) was dissolved in 100 ml of DMFand a 2 fold molar excess of N-hydroxysuccinimide was added understirring. A 1.1 fold molar excess of dicyclohexylcarbodiimide dissolvedin 10 ml of DMF was then added and the mixture was left overnight atroom temperature to produce the NHS ester (Compound X).

[0305] (e) Preparation of Compound XI (dUTP with Glycylglycine Linker)

[0306] 10 uMoles of allylamine dUTP were dissolved in 0.5 ml of 0.3MNaHCO₃ followed by addition of 15 umoles of Compound X from step (d) andincubation at room temperature for 2 hours to form Compound XI. LiAc wasthen added to a final concentration of 0.5M and the nucleotide product(Compound XI) was precipitated by addition of 5 volumes of ethanol andleaving the solution overnight at −20° C. The amine was deprotected bydissolving the precipitate in 1M LiOH for 1 hour at room temperature.The solution was neutralized with glacial acetic acid in the cold andthe Triphosphate was precipitated with ethanol as above.

[0307] (f) Preparation of Compound XII (dUTP with Tetraglycyl Linker)

[0308] Compound XI from step (e) was further treated by repeating step(e) to add an additional glycylglycine linker unit thereby forming5′-allylamido-(tetraglycyl) amine dUTP (Compound XII). The amine wasdeprotected and the Triphosphate precipitated as described above in step(e).

[0309] (g) Preparation of Compound XIII (Attachment of Aphenylic TAMRAAnalogue to dUTP)

[0310] 20 umoles of Compound XII from step (f) was dissolved in 2 ml ofNaHCO₃ (0.3 M) and LiCl (0.7 M) and cooled on ice. The active ester ofthe dye (40 umoles) in chloroform from step (b) (Compound VIII) wasdried in vacuum and dissolved in DMF (2 ml). This solution was thenadded to the ice cold dUTP solution and the mixture was stirred in thedark overnight at room temperature. The mixture was diluted with 20 mlwater and loaded onto a DEAE-Sephadex A₂₄ (20 ml) column at 4′ C. Thecolumn was washed with TEAC (0.1 M, pH 7.8, 50 ml) and the product waseluted with a linear gradient of 0.1-0.8 M TEAC, pH 7.8. The fractionsthat were pure by HPLC were combined. The TEAC was removed in vacuum byrepeated evacuations following the addition of water. The residue wasdissolved in lithium acetate (4 M) and precipitated with 4 volumes ofabsolute ethanol, then dissolved in water and stored at −70 to give 13.6mg of Compound XIII.

[0311] It should be noted that the example cited above used atetraglycyl rigid arm linker. The same methods that were described abovecould have been used to synthesize compounds with other lengths. Forinstance, compound XII (dUTP with tetraglycyl arm) from step (f) couldhave been manipulated further by a repetition of step (e) and addinganother glycylglycine unit therby creating a hexaglycyl arm. Similarly,the activation steps described for preparation of glycylglycine (steps cand d) could also been carried out with glycine as the starting materialthereby allowing addition of single glycyl units.

EXAMPLE 5 Preparation of a Labeled Nucleotide with a Rigid Linker Armand an Aphenylic Texas Red Analogue

[0312] (a) Preparation of Compound XIV(3,6-Bis-Julolidinoxanthen-9-Propionic Acid)

[0313] 8-Hydroxyjulolidine (10 g) and succinic anhydride (2.6 g) werecombined under argon and heated at 130° C. for 2 hours with stirring.The mixture was cooled, H₂O (150 ml) was added and the mixture wasrefluxed for 15 minutes and then cooled. The water layer was discardedand the glassy dark brown residue was dissolved by the addition of H₂Ofollowed by an adjustment to pH 10 with 1M NaOH while stirring. The pHof the solution was then brought down to 2 by the addition of 1M HCl atwhich point the product precipitated again. The mixture was centrifuged,the supernatant was discarded and the pellet was washed by suspending itin water and recentrifuging, The pellet was then lyophilized to give 3.6g of product (Compound XIV). The fluorescent spectrum of Compound XIV isshown in FIG. 8.

[0314] (b) Attachment of Label to a Nucleotide

[0315] Subsequent steps for the preparation of the active ester ofCompound XIV and attachment to dUTP with a rigid linker arm were carriedout as described in Example 4.

EXAMPLE 6 Preparation of Cyanine Dyes with a Rigid Linker Arm

[0316] (a) Preparation of Compound XV [(2,3,3, Trimethyl-3-H-indol-5-yl)Acetic Acid]

[0317] 110 g of 4-Hydrazinobenzoic acid were mixed with 450 ml ofglacial acetic acid and 250 ml of 3-methyl-2-butanone under stirring.The mixture was heated at 128° C.-130° C. for 6 hours and left to coolovernight at room temperature. The glacial acetic acid and3-methyl-2-butanone were removed under vacuum and the solid wastriturated with 300 ml H₂O, filtered and washed again with 300 ml H₂O.The cake was subsequently dried under vacuum. The solid was thenrecrystalized from ethyl acetate resulting in Compound XV.

[0318] (b) Preparation of Compound XVI (Diglycylallylamine)

[0319] Compound X from step (d) of Example 4 was reacted with a 1.2 foldexcess of allylamine acetate in a 50:50 mixture of DMF/H₂O. The solutionwas maintained at pH 8 by the addition of triethylamine and the reactionwas carried out for 4 hours at room temperature. The solution was driedunder vacuum and the mixture was triturated with H₂O to removetriethylamine salts. The slurry was filtered, washed with cold H₂O,lyophilized and dried resulting in Compound XVI

[0320] (c) Preparation of Compound XVII [(2,3,3 Trimethyl-3-H-indol-5yl)Acetamido Diglycylallylamine]

[0321] 20.6 g of Compound XV prepared in Example 7 were dissolved in 100ml of DMF followed by addition of 20 g of N-hydroxysuccinimide understirring. A mixture of 22 g of dicyclohexyl carbodiimide dissolved in 30ml of DMF was then added. The mixture was left at room temperatureovernight and the next day, urea was removed by filtration. 30 g ofCompound XVI from step (a) was dissolved in 100 ml of 50:50 mixture ofethanol and 1M LiOH in H₂O to liberate the amine. This solution wasneutralized with acetic acid to pH 8 and added to the filtrate above. Anequivalent amount of triethanolamine was slowly added to the solutionover a 1 hour period. The mixture was left at room temperature overnightand the resultant precipitate was filtered and extracted with 500 ml ofchloroform to produce Compound XVII.

[0322] (d) Preparation of Compound XVIII [(2,3,3Trimethyl-3-H-indol-5yl) Acetamido Diglycylailylamido EthylammoniumIodide]

[0323] Chloroform was removed from Compound XVII by vacuum. The glassyresidue was dissolved in 200 ml of DMF followed by removal of the DMF byvacuum. The residue was mixed with 150 ml dichlorobenzene and 100 mlethyliodide and refluxed at 16 hour at 100° C. After cooling, thesolvent was removed by decantation. The glassy residue was trituratedwith ether to produce Compound XVIII.

[0324] (e) Preparation of Cyanine Dyes and Cyanine Dye LabeledNucleotides

[0325] Compound XVIII was used without any further purification tosynthesize the cyanine dyes as described in Examples 1 and 2. Thestructure of a Cy 3 analogue made with Compound XVIII is given below.

[0326] The presence of the terminal alkene bond allowed labeling of dUTPas described in Example 3. When tested in a conventional cDNA synthesisassay, significantly higher incorporation was seen with dUTP labeledwith Compound XVIII as compared to a commercially available Cy-3 labeleddUTP (Cat. No. PA 530220) from Amersham Biosciences Corp., Piscataway,N.J.

EXAMPLE 7 meta-EthD, with and Without DNA

[0327] a) Synthesis of meta-EthD

[0328] The synthesis of meta-EthD was carried out according to themethod described by Kuhlmann et al., supra. A diagram of the syntheticsteps is given in FIG. 9. In this procedure, the2-amino-diphenyl-compound (1) was condensed with acid chloride (2) togive the amide (3) which was then converted to a cyclic form to give thephenanthridine (4). This compound (4) was hydrolyzed to give the acid(5), converted to the acid chloride (6) and then condensed with1,5-diamino-pentane to give the homodimer. The homodimer was methylatedto give (7) which was reduced to give the final product (8) meta-EthDwhose structure is given in FIG. 2.

[0329] b) Spectral Analysis

[0330] meta-EthD was excited at 493 nm and gave emission of 1×10⁵counts/second at 617 nm (FIG. 10A). When double stranded DNA was added,the emission increased to 6×10⁵ (FIG. 10B). In contrast, when excited ata wavelength of 350 nm, the emission at 600 nm was 2×10⁴ counts/secondthat increased to approximately 3.25×10⁶ counts/second upon the additionof DNA (FIG. 11).

EXAMPLE 8 Energy Transfer Between a Donor Nucleotide and an AcceptorNucleotide

[0331] The sequence of an amplicon that can be made from an HIVantisense construct is given in FIG. 12. A description of the derivationof this construct is given in Liu et al., (1997) J. Virol 71; 4079-4085.PCR of target analytes in a sample can be carried out in the presence ofa mixture of dUTP labeled with fluorescein as an energy donor and dUTPlabeled with Compound XIII from Example 4 as an energy acceptor usingthe primers shown in FIG. 12. During the course of amplification,nucleic acid strands are synthesized that incorporate each of theselabels. Illumination at a wavelength appropriate for fluoresceinfollowed by detection at a wavelength appropriate for the emission ofCompound XIII should result in signal generation whenever donornucleotides and acceptor nucleotides are in sufficient proximity. Eithersingle strands or double strands could be analyzed for this purpose.

EXAMPLE 9 Energy Transfer Between an Intercalator and an IncorporatedDye

[0332] PCR is carried out using the same primers as used in Example 8.However, in this example, the reaction is carried out in the presence ofSYBR Green and a labeled dUTP from Example 7. As incorporation proceeds,double-stranded DNA begins to accumulate that has Compound XVIII labelednucleotides incorporated. As described previously, SYBR Green displaysenhanced fluorescence after binding to double-stranded DNA. Since SYBRGreen maximally emits at 521 nm and Compound XVIII maximally absorbs at550 nm, fluorescence from Compound XVIII should increase as synthesisproceeds due to energy transfer form SYBR green donors to Compound XVIIIas an acceptor thus indicating successful amplification of targetsequences.

EXAMPLE 10 Energy Transfer Between an Intercalator and an IncorporatedDye with Primers that Comprise Quencher Moieties

[0333] This example is carried out as describe in Example 9 except thatthe primers are labeled with quenchers as follows:5′ CAU*GATCCGGAU*GGGAGGTG 3′ and 5′ GCACAU*CCGGAU*AGU*AGA 3′

[0334] where U* are uridine moieties modifed with a non-fluorescent3-amino xanthene as described by Singer and Haugland in U.S. Pat. No.6,323,337 that absorb at about 530 nm. PCR is carried out with theseprimers in the presence of a labeled dUTP from Example 7 and SYBR Greenas described above. Fluorescence from the intercalated SYBR Green can beabsorbed either by Compound XVIII or by the quencher. If Primer-dimersare formed, these comprise only primers and their complements. As suchenergy transfer should most efficiently take place with the quenchersand thereby reduce spurious signal generation from primer-dimersynthesis. On the other hand, amplicons derived from amplfication oftarget sequences have segments where only compound XVIII is insufficient proximity to the SYBR for energy transfer to take place andtarget dependent signals are generated as synthesis proceeds.

EXAMPLE 11 Energy Transfer Between a Probe and an IncorporatedNucleotide

[0335] PCR can be carried out with the same primers used in Example 8.In this reaction mixture, potential donors are supplied in the form ofdUTP labeled with Compound XVIII form Example 7. The reaction mixturealso contains a DNA probe labeled with Texas Red moieties that can actas energy acceptors. The probe has the sequence

[0336] 5′ U^(F)MTGGU^(F)GAGTATCCCU^(F)GCCTAACTCU^(F) 3′

[0337] where U^(F) indicates a Uridine labeled with Texas Red. Theposition of this probe in the amplicon is shown in FIG. 12. The probe isalso blocked at the 3′ end such that it is incapable of being extended.As amplification is carried out, hybridization of the probe to labeledamplicon strands allow energy transfer to take place between CompoundXVIII and Texas Red that should increase as more amplicon strands aregenerated.

EXAMPLE 12 Endonuclease Digestion and Strand Extension Using aHomopolymeric Target as a Substrate

[0338] The steps in this example are shown in FIG. 13. A CNAC with threesegments can be synthesized that has the sequence:

[0339] 5′-UUUUUUUUUUTTTTQQQQQQQQ-3′

[0340] where U is a uridine ribonucleotide, T is a thymidinedeoxyribonucleotide and Q is an inosine ribonucleotide and the 3′ endhas been modifed to prevent extension. In this example, theribonucleotides are 2′-O-methyl as described by Shibahara et al., (1987)Nucl. Acids Res.15; 4403-4415 and Baranov et al., (1997) Nucl. AcidsRes. 25; 2266-2273 (both of which are incorporated by reference). TheCNAC can be hybridized to a library of poly A mRNA (step A) forming:

[0341] a first complex with the oligo-uridine first segment bound to aportion of the poly A tail,

[0342] a second complex with the oligo-thymidine second segment bound toa second portion of the poly A tail; and

[0343] a third complex with the oligo-inosine third segment bound to athird portion of the poly A tails.

[0344] In this example, the first and third complexes will be resistantto the actions of RNase H and the second complex should form a substratefor RNAse activity since four deoxyribonucleotides are known to besufficient. Digestion with RNase H at 20-25° C. (step B) should inducecleavage in the poly A segment bound to the oligoT's in the secondcomplex and release of the cleaved poly A tail. Provision of dATP, dCTPand Reverse transcriptase allows extension of the 3′ end of the mRNA(step C). Additionally, if these reagents are present during the RNase Hdigestion step they may help stabilize the binding of the CNAC to the 3′end after endonucleolytic cleavage as described previously. It should benoted that although the inosine is capable of binding to the poly Asegment, when it is used as a template, it preferentially incorporatescytosine thereby introducing a new oligo-C segment into the end of themRNA. Removal of the CNAC allows the oligo-C segment to be used asprimer binding site for an oligonucleotide containing a complementaryoligo-G segment and an RNA promoter sequence. Synthesis of a cDNAstrand, production of a second cDNA strand and generation of a labeledlibrary can then be carried out by any method described previouslyincluding U.S. Pat. No. 5,891,636 and Rabbani et al. in U.S. patentapplication Ser. No. 09/896,897 filed Jun. 30, 2001.

EXAMPLE 13 Addition of an RNA Polymerase Sequence to an Analyte

[0345] This example is carried out as described in Example 1.2 exceptthat the third segment of the CNAC comprises unmodified ribonucleotidesand contains the sequence for an RNA promoter. As such, after strandextension in step (c), a new third complex is formed where the extendednucleotides are deoxyribonucleotides and the CNAC third segmentcomprises ribonucleotide. This is a substrate for RNase H digestionwhich can then be used to generate a single-stranded segment at the 3′end of the mRNA that is complementary to the RNA promoter sequence. Aprimer with promoter sequence can then be hybridized to the extendedsegment of the mRNA to synthesize a cDNA with a promoter at the 5′ end.Subsequent events can be carried out as described in Example 12. Theremaining portion of the CNAC can be removed prior to binding of theprimer or extension of the primer can allow a strand displacement event.

EXAMPLE 14 Preparation of a Dioxetane Derivative that is Capable ofLight Generation After an Enzyme Catalyzed Intrachain Rearrangement

[0346] A schematic of the steps that can be used to synthesize anintermediate compound for derivatization of a dioxetane is shown in FIG.14. The series of steps shown in this schematic can be carried out usingstandard chemistry methods. In the last step of this procedure, compound(e) can be attached to a dioxetane derivative where both “Q” and “Z” areas described previously. This dioxetane derivative (f) comprises an R1and an R2 group joined to adjacent sites of a cyclic ring as disclosedand defined in the present invention.

EXAMPLE 15 Potential Series of Enzyme Dependent Events with Compound (f)from Example 14

[0347] In the presence of Acylase I, a cleavage event takes place thatgenerates a free primary amine as shown by compound (f) being convertedto compound (g) in FIG. 15. Due to the proximity of the released primaryamine to the benzoyl residue in compound (g) and subsequent formation ofa six-membered ring in the transition state, compound (h), the internalrearrangement to produce compound (i) is a very rapid reaction. Thepresence of the phenoxy group in compound (i) makes it an unstabledioxetane that should generate light as it decomposes. It has beenpreviously described in the literature that a similar displacement cantake place with an acyl residue and a primary amine or thiol at the endof a chain attached to the acyl group. These previously describedreactions should not have the favorable kinetics of the reaction shownin this example.

[0348] This example shows an enzymatic reaction that converts R1 intoR1* thereby producing a reactive group G1 that is at the end of a chainattached to one site of a cyclic ring. In this particular example,Acylase I is the enzyme and G1 is a free primary amine. The reactioncontinues with G1 interacting with a benzoyl group (G2) that is attachedto a different site on the cyclic ring. This intermediate is shown as(h) in FIG. 15. An internal rearrangement takes place between the amineand benzoyl group (G1 and G2 respectively) leading to the intrachaintransfer of the benzoyl group and generation of an unstable lightemitting dioxetane.

[0349] Many obvious variations will no doubt be suggested to those ofordinary skill in the art in light of the above detailed description andexamples of the present invention. All such variations are fullyembraced by the scope and spirit of the invention as more particularlydefined in the claims that now follow.

1 12 1 20 DNA Artificial Sequence Description of Artificial SequencePrimer 1 caugatccgg augggaggtg 20 2 18 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 gcacauccgg auaguaga 18 3 27DNA Artificial Sequence Description of Artificial Sequence Syntheticprobe sequence 3 uaatggugag tatcccugcc taactcu 27 4 22 DNA ArtificialSequence Description of Artificial Sequence Synthetic chimeric nucleicacid construct sequence 4 uuuuuuuuuu ttttnnnnnn nn 22 5 33 DNAArtificial Sequence Description of Artificial Sequence Primer 5gcgacctgcg aatgctatgg atcaggctag cca 33 6 20 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 catgatccgg atgggaggtg 20 727 DNA Artificial Sequence Description of Artificial Sequence Syntheticprobe 7 taatggtgag tatccctgcc taactct 27 8 78 DNA Human immunodeficiencyvirus 8 catgatccgg atgggaggtg ggtctgaaac gataatggtg agtatccctgcctaactcta 60 ttcactatcc ggatgtgc 78 9 22 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 gcacatccgg atagtgaata ga 2210 65 RNA Artificial Sequence Description of Artificial Sequence Primer10 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60aaaaa 65 11 14 RNA Artificial Sequence Description of ArtificialSequence Primer 11 aaaaaaaaaa aaaa 14 12 26 RNA Artificial SequenceDescription of Artificial Sequence Primer 12 aaaaaaaaaa aaaaaaaacccccccc 26

1-286. (Canceled)
 287. A dye composition of the formula R-FluorescentDye wherein R is covalently linked to said Fluorescent Dye comprises twoor more members in combination from a) unsaturated aliphatic groups; b)unsaturated heterocyclic groups; c) aromatic groups; and wherein R iscapable of providing a conjugated system or an electron delocalizedsystem with said fluorescent dye.
 288. The dye composition of claim 287,wherein said unsaturated aliphatic groups comprise an alkene or analkyne.
 289. The dye composition of claim 287, wherein said aromaticgroups comprise a phenyl group, an aryl group or an aromaticheterocyclic group.
 290. The dye composition of claims 288 or 289,wherein said unsaturated aliphatic groups or aromatic groups aresubstituted.
 291. The dye composition of claim 290, wherein saidsubstituted unsaturated aliphatic groups or substituted aromatic groupscomprise alkyl groups, aryl groups, alkoxy groups, phenoxy groups,amines, amino groups, amido groups, carboxy groups, nitrates, nitrites,sulfonates, sulfhydryl groups or phosphates.
 292. The dye composition ofclaim 290, wherein said substituted aromatic groups comprise a fusedring structure.
 293. The dye composition of claim 292, wherein saidfused ring structure is a naphthalene, anthracene or a phenanthrene.294. The dye composition of claim 287, wherein said combinationcomprises two members of the same group or of different groups.
 295. Thedye composition of claim 294, wherein said different groups comprise anunsaturated aliphatic group (a) and an unsaturated heterocyclic group(b); an unsaturated aliphatic group (a) and an aromatic group (c); or anunsaturated heterocyclic group (b) and an aromatic group (c).
 296. Thedye composition of claim 287, wherein said fluorescent dye comprises ananthracene, a xanthene, a cyanine, a porphyrin, a coumarin or acomposite dye.
 297. The dye composition of claim 287, further comprisinga charged or polar R′ group.
 298. The dye composition of claim 297,wherein said charged or polar R′ group increases aqueous solubility ofsaid composition.
 299. The dye composition of claim 288 or 297, furthercomprising a reactive group R_(x) attached to either said fluorescentdye, said R group or said R′ group.
 300. The dye composition of claim299, further comprising a linker arm attaching said reactive group R_(x)to said fluorescent dye, said R group or said R′ group.
 301. The dyecomposition of claim 299, wherein said reactive group R_(x) comprisessulfhydryl, hydroxyl, amine, isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono- or di-halogen substitutedpyridine, mono- or di-halogen substituted diazine, maleimide, aziridine,sulfonylhalide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde.
 302. Thedye composition of claim 300, wherein said reactive group R_(x)comprises sulfhydryl, hydroxyl, amine, isothiocyanate, isocyanate,monochlorotriazine, dichlorotriazine, mono- or di-halogen substitutedpyridine, mono- or di-halogen substituted diazine, maleimide, aziridine,sulfonylhalide, acid halide, hydroxysuccinimide ester,hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde.
 303. Thedye composition of claim 299, wherein as a reactive group R is capableof forming a carbon-carbon linkage with a target.
 304. The dyecomposition of claim 300, wherein as a reactive group R is capable offorming a carbon-carbon linkage with a target.
 305. The dye compositionof claim 303, wherein said reactive group R comprises an alkene group,an alkyne group, a halogenated compound or a metallo-organic compound.306. The dye composition of claim 304, wherein said reactive group Rcomprises an alkene group, an alkyne group, a halogenated compound or ametallo-organic compound.
 307. A labeled target having the structure[R-Dye]—target wherein said Dye is a fluorescent dye, wherein R iscovalently linked to said Dye, and wherein R comprises two or moremembers in combination from a) unsaturated aliphatic groups; b)unsaturated heterocyclic groups; c) aromatic groups; and wherein R iscapable of providing a conjugated system or an electron delocalizedsystem with said Dye. comprises two or more unsaturated aliphaticgroups, unsaturated heterocyclic groups, aromatic groups, orcombinations of the foregoing groups and wherein R is covalentlyattached to said fluorescent dye and is capable of providing aconjugated system or an electron delocalized system with saidfluorescent dye, and wherein said target is covalently attached to saidDye or said R.
 308. The labeled target of claim 307, wherein saidunsaturated aliphatic groups comprise an alkene or an alkyne.
 309. Thelabeled target of claim 307, wherein said aromatic groups comprise aphenyl group, an aryl group or an aromatic heterocyclic group.
 310. Thelabeled target of claims 308 or 309, wherein said unsaturated aliphaticgroups or aromatic groups are substituted.
 311. The labeled target ofclaim 307, wherein said substituted unsaturated aliphatic groups orsubstituted aromatic groups comprise alkyl groups, aryl groups, alkoxygroups, phenoxy groups, amines, amino groups, amido groups, carboxygroups, nitrates, nitrites, sulfonates, sulfhydryl groups or phosphates.312. The labeled target of claim 310, wherein said substituted aromaticgroups comprise a fused ring structure.
 313. The labeled target of claim312, wherein said fused ring structure is a naphthalene, anthracene or aphenanthrene.
 314. The labeled target of claim 307, wherein saidcombination comprises two members of the same group or of differentgroups.
 315. The labeled target of claim 314, wherein said differentgroups comprise an unsaturated aliphatic group (a) and an unsaturatedheterocyclic group (b); an unsaturated aliphatic group (a) and anaromatic group (c); or an unsaturated heterocyclic group (b) and anaromatic group (c).
 316. The labeled target of claim 307, wherein saidfluorescent dye comprises an anthracene, a xanthene, a cyanine, aporphyrin, a coumarin or a composite dye.
 317. The labeled target ofclaim 307, further comprising a charged or polar R′ group.
 318. Thelabeled target of claim 307, wherein said charged or polar R′ groupincreases aqueous solubility of said composition.
 319. The labeledtarget of claim 317, further comprising a linker arm attaching saidtarget to said fluorescent dye, said R group or said R′ group.
 320. Thelabeled target of claim 307, wherein said target comprises a protein, apeptide, a nucleic acid, a nucleotide or a nucleotide analog, areceptor, a natural or synthetic drug, a synthetic oligomer, a syntheticpolymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, anantigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- orpolysaccharide.
 321. The labeled target of claim 320, wherein saidligand comprises biotin, iminobiotin, digoxygenin or fluorescein, andthe dye comprises a fluorescent dye.